Pharmaceutical products from fungal strains

ABSTRACT

Disclosed herein are compositions comprising an isolated cellulose degrading fungus and pharmaceutical substances produced by the fungus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a nonprovisional application of and claims priority35 U.S.C. §119(e) to U.S. Provisional Application No. 61/372,828, filedAug. 11, 2010; U.S. Provisional Application No. 61/411,860, filed Nov.9, 2010; U.S. Provisional Application No. 61/475,176, filed Apr. 13,2011; and under 35 U.S.C. §365(b) to PCT Application No.PCT/US2011/035644, filed May 6, 2011, each of which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledMENON007WO.TXT, created Aug. 10, 2011, which is 3.11 Kb in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present application generally relates to the use of microbial andchemical systems to produce antibiotic and other pharmaceuticalsubstances.

BACKGROUND

Petroleum is facing declining global reserves and contributes to morethan 30% of greenhouse gas emissions driving global warming. Annually800 billion barrels of transportation fuel are consumed globally. Dieseland jet fuels account for greater than 50% of global transportationfuels.

Significant legislation has been passed, requiring fuel producers to capor reduce the carbon emissions from the production and use oftransportation fuels. Fuel producers are seeking substantially similar,low net carbon fuels that can be blended and distributed throughexisting infrastructure (e.g., refineries, pipelines, tankers).

Due to increasing petroleum costs and reliance on petrochemicalfeedstocks, the chemicals industry is also looking for ways to improvemargin and price stability, while reducing its environmental footprint.The chemicals industry is striving to develop greener products that aremore energy, water, and CO₂ efficient than current products. Fuelsproduced from biological sources, such as biomass, represent one aspectof the process.

SUMMARY OF THE INVENTION

A system and method are provided which utilize microbes to convertbiomass feedstock into fuel. In one aspect of the invention, a method ofproducing aromatic compounds, animal feed, and lipids includes receivinga feedstock including biological matter; separating the feedstock into aliquid phase feedstock and a solid phase feedstock; adding water andnutrients to the solid phase feedstock, thereby producing a liquidculture; inoculating the liquid culture with one or more microbescapable of converting the solid phase feedstock into aromatic compoundsand lipids, the inoculated liquid culture yielding microbial biomass;providing suitable conditions for the microbes to convert the solidphase feedstock to aromatic compounds and lipids; and extractingproduced aromatic compounds and lipids. Lipids are chemically convertedto alkanes and related hydrocarbons for fuel, or alternatively aretransesterified to produce biodiesel, both using processes known in theart.

In some aspects, the liquid feedstock and the solid feedstock are notseparated prior to inoculation of the culture. In some aspects, thesolid phase feedstock is pretreated to release cellulosic and/orhemicellulosic breakdown products, such as oligosaccharides, into theliquid medium. In other embodiments, the solid phase feedstock need notbe pretreated.

In another aspect of the invention, the liquid phase feedstock,representing hydrolyzates of the cellulosic and/or hemicellulosicportions of the biological matter, is inoculated with microbes capableof converting the hydrolyzates into lipids. Lipids are chemicallyconverted to alkanes and related hydrocarbons for fuel, or alternativelyare transesterified to produce biodiesel, both using processes known inthe art.

In another aspect of the invention, a system for producing fuelcomponents includes a bioreactor having an inoculated liquid cultureincluding microbes and biomass; and a controller in communication withthe bioreactor, the controller providing operating instructions to thebioreactor; and where the bioreactor yields the fuel components.

In yet another aspect of the invention, a method of producing aromaticcompounds, animal feed, and lipids includes receiving a feedstockincluding biological matter; adding water and nutrients to thefeedstock; inoculating the feedstock with microbes capable of convertinga portion of the feedstock into aromatic compounds and lipids; providingsuitable conditions for the microbes to convert a portion of thefeedstock to aromatic compounds and lipids; and extracting producedaromatic compounds and lipids.

Also described herein are compositions and processes that comprise orotherwise utilize a cellulose degrading fungus of the genus Penicillium.

Some aspects of the compositions described herein relate to an isolatedcellulose degrading fungus of the genus Penicillium. In some suchaspects, the isolated cellulose degrading fungus of the genusPenicillium comprises a fungus that is the same species as the fungushaving NRRL deposit Accession No: 50410. In other aspects, the isolatedcellulose degrading fungus of the genus Penicillium comprises anisolated fungus comprising a 5.8 S ribosomal RNA gene sequence having atleast 98% nucleotide sequencing identity with the nucleic acid of SEQ IDNO: 1. In still another aspect, the isolated cellulose degrading fungusof the genus Penicillium further comprises an ITS1 sequence with atleast 98% sequence identity to SEQ ID NO: 2. In yet another aspect, theisolated cellulose degrading fungus of the genus Penicillium furthercomprises an ITS2 sequence with at least 98% sequence identity to SEQ IDNO: 3. In still another aspect, the isolated cellulose degrading fungusof the genus Penicillium further comprises a 28 S ribosomal RNA genesequence with at least 98% sequence identity to SEQ ID NO: 4. In yetanother aspect, the isolated cellulose degrading fungus of the genusPenicillium comprises Penicillium menonorum.

Also described herein is a living, in vitro culture of a cellulosedegrading fungus of the genus Penicillium comprising a nutrient medium,wherein the fungus is growing on the medium. In certain aspects, themedium is a solid medium. In other aspects, the medium is a liquidmedium. In certain aspects, the medium comprises a carbon source and anitrogen source, wherein the ratio of carbon to nitrogen in the mediumranges from about 1:1 to about 1000:1. In other aspects, the ratio ofcarbon to nitrogen in the medium ranges from about 20:1 to about 100:1.In still other aspects, the ratio of carbon to nitrogen in the medium isgreater than about 100:1.

In some aspects, the carbon source comprises one or more simple sugars.In one such aspect, the carbon source comprises cane juice and/or itscondensates including, but not limited to, dry solid obtained bycomplete evaporation.

In other aspects, the carbon source comprises a cellulosic carbon sourceor a lysate thereof. In a preferred aspect, the cellulosic carbon sourcecomprises a hydrolysate of a cellulosic carbon. In certain aspects, thecarbon source comprises or is derived from a material selected from thegroup consisting of grains, stover, forage, grasses, oilseed crops, nutshells, nut hulls, fruit pomace, plant waste, algae, wood, woodbyproducts, bark, paper, paper products, animal manure and food waste.In preferred aspects, the carbon source comprises or is derived from amaterial selected from the group consisting of stover of grains, stoverof oilseed crops, almond hulls, grape pomace, agriculture waste, yardwaste, wood chips, sawdust, the manure of herbivorous animals and foodwaste of plant origin. In other aspects, the cellulosic carbon sourcecomprises a lignocellulosic carbon source or a lysate thereof. In stillother aspects, the cellulosic carbon source comprises a hemicellulosiccarbon source or a lysate thereof.

Also described herein is a bioprocess reactor or bioreactor comprising acellulose degrading fungus of the genus Penicillium growing in a liquidmedium, wherein the fungus comprises at least 5% triacylglyceride(“TAG”) by weight. In a preferred aspect, the weight is dry weight. Insome aspects, the cellulose degrading fungus of the genus Penicilliumcomprises at least about 10% to about 50% triacylglyceride by dryweight. In a preferred aspect, the cellulose degrading fungus of thegenus Penicillium comprises at least about 25% to about 50%triacylglyceride by dry weight. In some aspects, the liquid methodcomprises a carbon source.

In some aspects, the carbon source comprises one or more simple sugars.In one such aspect, the carbon source comprises cane juice and/or itscondensates including, but not limited to, dry solid obtained bycomplete evaporation.

In other aspects, the carbon source comprises a cellulosic carbon sourceor a lysate thereof. In a preferred aspect, the cellulosic carbon sourcecomprises a hydrolysate of a cellulosic carbon. In certain aspects, thecarbon source comprises or is derived from a material selected from thegroup consisting of grains, stover, forage, grasses, oilseed crops, nutshells, nut hulls, fruit pomace, plant waste, algae, wood, woodbyproducts, bark, paper, paper products, animal manure and food waste.In preferred aspects, the carbon source comprises or is derived from amaterial selected from the group consisting of stover of grains, stoverof oilseed crops, almond hulls, grape pomace, agriculture waste, yardwaste, wood chips, sawdust, the manure of herbivorous animals and foodwaste of plant origin. In other aspects, the cellulosic carbon sourcecomprises a lignocellulosic carbon source or a lysate thereof. In stillother aspects, the cellulosic carbon source comprises a hemicellulosiccarbon source or a lysate thereof.

Also described herein is a bioprocess reactor or bioreactor comprising acellulose degrading fungus of the genus Penicillium growing in a liquidmedium, wherein the liquid medium comprises a carbon source and anitrogen source. In some aspects, the ratio of carbon to nitrogen in themedium ranges from about 1:1 to about 1000:1. In some aspects, the ratioof carbon to nitrogen in the medium ranges from about 20:1 to about100:1. In some aspects, the ratio of carbon to nitrogen in the medium isgreater than about 100:1.

In some aspects, the carbon source comprises one or more simple sugars.In one such aspect, the carbon source comprises cane juice and/or itscondensates including, but not limited to, dry solid obtained bycomplete evaporation.

In other aspects, the carbon source comprises a cellulosic carbon sourceor a lysate thereof. In a preferred aspect, the cellulosic carbon sourcecomprises a hydrolysate of a cellulosic carbon. In certain aspects, thecarbon source comprises or is derived from a material selected from thegroup consisting of grains, stover, forage, grasses, oilseed crops, nutshells, nut hulls, fruit pomace, plant waste, algae, wood, woodbyproducts, bark, paper, paper products, animal manure and food waste.In preferred aspects, the carbon source comprises or is derived from amaterial selected from the group consisting of stover of grains, stoverof oilseed crops, almond hulls, grape pomace, agriculture waste, yardwaste, wood chips, sawdust, the manure of herbivorous animals and foodwaste of plant origin. In other aspects, the cellulosic carbon sourcecomprises a lignocellulosic carbon source or a lysate thereof. In stillother aspects, the cellulosic carbon source comprises a hemicellulosiccarbon source or a lysate thereof.

In certain aspects, the liquid medium comprises dissolved oxygen in arange of about 0.1 mg/L to about 100 mg/L. In certain aspects, thedissolved oxygen is in a range of about 0.5 mg/L to about 40 mg/L.

Also described herein is a bioprocess reactor or bioreactor comprising acellulosic carbon source having a cellulose degrading fungus of thegenus Penicillium growing thereon, said cellulosic carbon source or alysate thereof present in a liquid medium.

In other aspects, the carbon source comprises a cellulosic carbon sourceor a lysate thereof. In a preferred aspect, the cellulosic carbon sourcecomprises a hydrolysate of a cellulosic carbon. In certain aspects, thecarbon source comprises or is derived from a material selected from thegroup consisting of grains, stover, forage, grasses, oilseed crops, nutshells, nut hulls, fruit pomace, plant waste, algae, wood, woodbyproducts, bark, paper, paper products, animal manure and food waste.In preferred aspects, the carbon source comprises or is derived from amaterial selected from the group consisting of stover of grains, stoverof oilseed crops, almond hulls, grape pomace, agriculture waste, yardwaste, wood chips, sawdust, the manure of herbivorous animals and foodwaste of plant origin. In other aspects, the cellulosic carbon sourcecomprises a lignocellulosic carbon source or a lysate thereof. In stillother aspects, the cellulosic carbon source comprises a hemicellulosiccarbon source or a lysate thereof.

Also described herein is a bioprocess reactor or bioreactor comprising acellulose degrading fungus of the genus Penicillium growing in a liquidmedium, said liquid medium comprising at least one lipid selected fromthe group consisting of triacylglyceride and phospholipids. In someaspects, the liquid medium comprises a carbon source and a nitrogensource, wherein the ratio of carbon to nitrogen in the medium rangesfrom about 1:1 to about 1000:1. In some aspects, the ratio of carbon tonitrogen in the medium ranges from about 20:1 to about 100:1. In someaspects, the ratio of carbon to nitrogen in the medium is greater thanabout 100:1.

In some aspects, the carbon source comprises one or more simple sugars.In one such aspect, the carbon source comprises cane juice and/or itscondensates including, but not limited to, dry solid obtained bycomplete evaporation.

In other aspects, the carbon source comprises a cellulosic carbon sourceor a lysate thereof. In a preferred aspect, the cellulosic carbon sourcecomprises a hydrolysate of a cellulosic carbon. In certain aspects, thecarbon source comprises or is derived from a material selected from thegroup consisting of grains, stover, forage, grasses, oilseed crops, nutshells, nut hulls, fruit pomace, plant waste, algae, wood, woodbyproducts, bark, paper, paper products, animal manure and food waste.In preferred aspects, the carbon source comprises or is derived from amaterial selected from the group consisting of stover of grains, stoverof oilseed crops, almond hulls, grape pomace, agriculture waste, yardwaste, wood chips, sawdust, the manure of herbivorous animals and foodwaste of plant origin. In other aspects, the cellulosic carbon sourcecomprises a lignocellulosic carbon source or a lysate thereof. In stillother aspects, the cellulosic carbon source comprises a hemicellulosiccarbon source or a lysate thereof.

In some aspects, the bioprocess reactor and bioreactor compositionsdescribed above can further comprise an impeller rotating at a stir rateof about 1 RPM to about 1200 RPM. In some aspects the stir rate can beabout 1 RPM to about 300 RPM. In some aspects the stir rate can be about20 to about 100 RPM. In other aspects, the stir rate can be about 60-70RPM.

Also described herein is a fungal culture in a vessel, wherein theculture comprises a liquid medium comprising a cellulose degradingfungus of the genus Penicillium in growth phase, a carbon source, anon-carbon nutrient mix, and MgSO₄ and/or other salt(s) of Mg at aconcentration of at least 0.5 mM. In certain aspects, the culture isapproximately 0, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240 orgreater than 240 hours old. In some aspects, the ratio of carbon tonitrogen in the medium ranges from about 1:1 to about 1000:1. In someaspects, the ratio of carbon to nitrogen in the medium ranges from about20:1 to about 100:1. In some aspects, the ratio of carbon to nitrogen inthe medium is greater than about 100:1.

In some aspects, the carbon source comprises one or more simple sugars.In one such aspect, the carbon source comprises cane juice and/or itscondensates including, but not limited to, dry solid obtained bycomplete evaporation.

In other aspects, the carbon source comprises a cellulosic carbon sourceor a lysate thereof. In a preferred aspect, the cellulosic carbon sourcecomprises a hydrolysate of a cellulosic carbon. In certain aspects, thecarbon source comprises or is derived from a material selected from thegroup consisting of grains, stover, forage, grasses, oilseed crops, nutshells, nut hulls, fruit pomace, plant waste, algae, wood, woodbyproducts, bark, paper, paper products, animal manure and food waste.In preferred aspects, the carbon source comprises or is derived from amaterial selected from the group consisting of stover of grains, stoverof oilseed crops, almond hulls, grape pomace, agriculture waste, yardwaste, wood chips, sawdust, the manure of herbivorous animals and foodwaste of plant origin. In other aspects, the cellulosic carbon sourcecomprises a lignocellulosic carbon source or a lysate thereof. In stillother aspects, the cellulosic carbon source comprises a hemicellulosiccarbon source or a lysate thereof.

Also described herein is a filtration apparatus comprising a filterhousing with a filtration surface disposed therein, and a mycelial matof a cellulose degrading fungus of the genus Penicillium dispersed onsaid filtration surface. In some aspects, the mycelial mat comprises amoisture content of less than about 50% w/w. In other aspects, themycelial mat comprises a moisture content of less than about 25% w/w. Instill other aspects, the mycelial mat comprises a moisture content ofless than about 15% w/w. In yet other aspects, the mycelial matcomprises a moisture content of less than about 5% w/w.

Also described herein is an extraction apparatus comprising a vesselcomprising a solvent and a fungus, wherein the solvent is in contactwith the fungus. In a preferred embodiment, the extraction apparatuscomprises hyphal filaments and/or other tissues or cellular componentsof a cellulose degrading fungus of the genus Penicillium dispersed in anaprotic solvent.

Also described herein is an extraction apparatus comprising a vesselcomprising a solvent and a fungus, wherein the solvent is in contactwith the fungus. In a preferred embodiment, the extraction apparatuscomprises hyphal filaments and/or other tissues or cellular componentsof a cellulose degrading fungus of the genus Penicillium dispersed in asolvent comprising less than 80% water. In some aspects, the solventcomprises at least one aliphatic solvent. In certain aspects, thesolvent comprises at least one polar solvent. In certain aspects, thepolar solvent is aprotic. In a preferred aspect, the solvent compriseshexane and ethanol. In some aspects, the ethanol is at a concentrationof about 5-25%. In some aspects, the ethanol is at a concentration ofabout 10-20%.

Also described herein is a bioprocessing facility for producingco-products, the facility comprising a plurality of bioprocess reactorscomprising: a cellulose degrading fungus of the genus Penicilliumgrowing in a liquid medium. In some aspects, the liquid medium comprisesa carbon source and a nitrogen source, wherein the ratio of carbon tonitrogen in the medium ranges from about 1:1 to about 1000:1. In someaspects, the ratio of carbon to nitrogen in the medium ranges from about20:1 to about 100:1. In some aspects, the ratio of carbon to nitrogen inthe medium is greater than about 100:1.

In some aspects, the cellulose degrading fungus of the genus Penicilliumcomprises at least 5% triacylglyceride by weight. In a preferred aspect,the weight is dry weight. In some aspects, the cellulose degradingfungus of the genus Penicillium comprises at least about 10% to about50% triacylglyceride by dry weight. In a preferred aspect, the cellulosedegrading fungus of the genus Penicillium comprises at least about 25%to about 50% triacylglyceride by dry weight.

In some aspects, the carbon source comprises one or more simple sugars.In one such aspect, the carbon source comprises cane juice and/or itscondensates including, but not limited to, dry solid obtained bycomplete evaporation.

In other aspects, the carbon source comprises a cellulosic carbon sourceor a lysate thereof. In a preferred aspect, the cellulosic carbon sourcecomprises a hydrolysate of a cellulosic carbon. In certain aspects, thecarbon source comprises or is derived from a material selected from thegroup consisting of grains, stover, forage, grasses, oilseed crops, nutshells, nut hulls, fruit pomace, plant waste, algae, wood, woodbyproducts, bark, paper, paper products, animal manure and food waste.In preferred aspects, the carbon source comprises or is derived from amaterial selected from the group consisting of stover of grains, stoverof oilseed crops, almond hulls, grape pomace, agriculture waste, yardwaste, wood chips, sawdust, the manure of herbivorous animals and foodwaste of plant origin. In other aspects, the cellulosic carbon sourcecomprises a lignocellulosic carbon source or a lysate thereof. In stillother aspects, the cellulosic carbon source comprises a hemicellulosiccarbon source or a lysate thereof.

In some aspects of the above-described bioprocessing facilities, one ormore bioprocess reactors can further comprise an impeller rotating at astir rate of about 1 RPM to about 1200 RPM. In some aspects the stirrate can be about 1 RPM to about 300 RPM. In some aspects the stir ratecan be about 20 to about 100 RPM. In other aspects, the stir rate can beabout 60-70 RPM.

Also described herein is a process for manufacturing a plurality ofco-products, the process comprising: a) inoculating a bioprocess reactorwith an inoculum of a cellulose degrading fungus of the genusPenicillium; b) allowing a sufficient time for production of TAG by saidfungus, wherein said TAG comprises at least 5% of the dry weight of thefungus; c) harvesting said fungus; d) extracting lipids from saidfungus, thereby producing a lipid co-product. The process can furthercomprise e) drying said fungus, thereby producing a feed co-product. Insome aspects, the TAG comprises at least 35% of the dry weight of thefungus. In other aspects, the lipid co-product comprises a TAGco-product and a phospholipid co-product. In still other aspects, thephospholipid co-product comprises lecithin. In yet other aspects, thefeed co-product is suitable for animal and/or human consumption. Incertain preferred aspects, the fungus comprises Penicillium menonorum.In preferred aspects, the fungus comprises a species being the same asNRRL deposit Accession No: 50410.

Also described herein is a process for producing a harvested fungus anda conditioned liquid medium, the process comprising: a) incubating acellulose degrading fungus of the genus Penicillium in a liquid mediumfor a sufficient time for production of TAG by said fungus; and b)separating said fungus from said liquid medium, thereby producing aharvested fungus and a conditioned liquid medium. In some aspects, theliquid medium comprises a carbon source and a nitrogen source, whereinthe ratio of carbon to nitrogen in the medium ranges from about 1:1 toabout 1000:1. In some aspects, the ratio of carbon to nitrogen in themedium ranges from about 20:1 to about 100:1. In some aspects, the ratioof carbon to nitrogen in the medium is greater than about 100:1. Incertain aspects, the TAG content in said harvested fungus comprises atleast 5% of the dry weight of the fungus. In some aspects, the TAGcomprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least50% of the dry weight of the fungus. In certain aspects, the harvestedfungus comprises an enriched Fe content. In some aspects, the harvestedfungus comprises an enriched Ni content. In some aspects, the harvestedfungus comprises an enriched malic acid content. In some aspects, theharvested fungus comprises an enriched content of one or more aminoacids selected from the group consisting of: L-isoleucine, L-tryptophan,and L-serine. In certain preferred aspects, the cellulose degradingfungus comprises Penicillium menonorum. In preferred aspects, the funguscomprises a species being the same as NRRL deposit Accession No: 50410.

Also described herein is a conditioned liquid medium produced by theprocess described hereinabove. In certain aspects, the conditionedliquid medium comprises a carbon source and a nitrogen source, whereinthe ratio of carbon to nitrogen in the medium ranges from about 1:1 toabout 1000:1. In some aspects, the ratio of carbon to nitrogen in themedium ranges from about 20:1 to about 100:1. In some aspects, the ratioof carbon to nitrogen in the medium is greater than about 100:1. Incertain preferred aspects, the conditioned liquid medium has beenconditioned by a cellulose degrading fungus comprises Penicilliummenonorum. In preferred aspects, the fungus comprises a species beingthe same as NRRL deposit Accession No: 50410. In some aspects, theconditioned liquid medium comprises a nutrient mixture comprisingincreased iron (Fe) content. In certain preferred embodiments, thenutrient mixture comprises a concentration of between 0.1 mM and 10 mMof iron per every 40 g/L of carbon source. In some aspects, theconditioned liquid medium can comprise a nutrient mixture that comprisesNi. In certain aspects, NiSO₄ is added in a concentration ranging fromabout 1 ppm to about 20 ppm of NiSO₄. In some aspects, the conditionedliquid medium can comprise a nutrient mixture that comprises malic acid.In certain aspects, malic acid is at a concentration of about 0.01%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, or about 10%. In some aspects, the conditionedliquid medium can comprise a nutrient mixture that comprises one or moreamino acids selected from the group consisting of: L-isoleucine,L-tryptophan, and L-serine. In certain aspects, the one or more aminoacids is at a concentration of about 1 mg/L, 10 mg/L, 50 mg/L, 100 mg/L,200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L,900 mg/L, or about 1000 mg/L. In certain aspects, the one or more aminoacids is at a concentration of about 400 mg/L.

The conditioned media described herein can be used to support the growthand/or maintenance of various organisms, including organisms capable ofdegrading cellulose or partially degraded cellulose.

Also described herein is a facility comprising: a vessel for holdingliquid medium conditioned by a cellulose degrading fungus of the genusPenicillium; an extraction apparatus comprising an extract of lipidsderived from said fungus; and a drying apparatus comprising driedfungus. In some aspects, the extract of lipids comprises TAG. In otheraspects, the extract of lipids comprises phosphlipids. In other aspects,the phospholipids comprise lecithin. In yet other aspects, the driedfungus is suitable for animal and/or human consumption. In certainpreferred aspects, the fungus comprises Penicillium menonorum. Inpreferred aspects, the fungus comprises a species being the same as NRRLdeposit Accession No: 50410.

With respect to any of the above-described compositions and/orprocesses, the cellulose degrading fungus of the genus Penicillium canbe selected from the group consisting of a fungus of the genusPenicillium that is the same species as the fungus having NRRL depositAccession No: 50410; a fungus comprising a 5.8 S ribosomal RNA genesequence having at least 98% nucleotide sequencing identity with thenucleic acid of SEQ ID NO: 1; fungus comprising an ITS1 sequence with atleast 98% sequence identity to SEQ ID NO: 2; a fungus comprising an ITS2sequence with at least 98% sequence identity to SEQ ID NO: 3; a funguscomprising a 28 S ribosomal RNA gene sequence with at least 98% sequenceidentity to SEQ ID NO: 4; and Penicillium menonorum.

In some aspects of the above-described compositions and/or processesthat include a medium, the medium can comprise at least one dissolvednutrient selected from the group consisting of glycerol, yeast extract,corn steep, mycelial cell extract from earlier cultures of the fungus,sulfates, nitrates, calcium salts, ammonium phosphate, magnesiumsulfate, calcium chloride, ferric citrate, potassium sulfate, sodiumacetate, sodium molybdate, copper sulfate, cobalt nitrate, zinc sulfate,boric acid and manganese chloride. In certain preferred aspects, themedium comprises MgSO₄ and/or other salt(s) of Mg at a concentration ofat least 0.5 mM. In other aspects, the medium comprises a detergent. Insome such aspects, the medium comprises a non-ionic detergent selectedfrom Polysorbate 80 (Tween80), Polysorbate 20 (Tween20), deoxycholateand Brij-35. In certain aspects, the non-ionic detergent comprisesPolysorbate 80 (Tween80). In a preferred aspect, the liquid mediumcomprises Polysorbate 80 (Tween80) at a concentration of at least 0.01%.In some aspects, the medium is at a pH of at least about 3.5.

In some aspects of the above-described compositions and/or processesthat include a liquid medium, the liquid medium can comprise dissolvedoxygen in a range of about 0.1 mg/L to about 100 mg/L. In other aspects,the dissolved oxygen is in a range of about 0.5 mg/L to about 40 mg/L.

In some aspects of the above-described compositions and/or processesthat include a liquid medium, the liquid medium can comprise nutrientmixtures that provide for enhanced TAG accumulation in cultures offungal microbes. In some aspects, the nutrient mixture comprisesincreased iron (Fe) content. In certain aspects, the nutrient mixturecomprises increased iron content wherein the iron content in thenutrient mixture is increased by a factor of about two to a factor ofabout thirty. In certain embodiments, the nutrient mixture comprises aconcentration of about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM,0.7 mM, 0.8 mM, 0.9 mM to about 1 mM of iron per every 40 g/L of carbonsource. In certain preferred embodiments, the nutrient mixture comprisesa concentration of about 0.5 mM of iron per every 40 g/L of carbonsource.

In some aspects of the above-described compositions and/or processesthat include a liquid medium, the liquid medium can comprise a nutrientmixture that comprises Ni. In certain aspects, NiSO₄ is added in aconcentration ranging from about 0.1 ppm, 1 ppm, 10 ppm, 100 ppm toabout 1000 ppm. In certain aspects, NiSO₄ is added in a concentrationranging from about 1 ppm to about 100 ppm of NiSO₄. In certain aspects,NiSO₄ is added in a concentration ranging from about 1.1 ppm to about11.1 ppm of NiSO₄.

In some aspects of the above-described compositions and/or processesthat include a liquid medium, the liquid medium can comprise a nutrientmixture that comprises malic acid. In certain aspects, malic acid isadded to the nutrient mixture before the culture is inoculated. Incertain aspects, the malic acid concentration in the nutrient mix priorto inoculation is about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or about 10%.

In some aspects of the above-described compositions and/or processesthat include a liquid medium, the liquid medium can comprise a nutrientmixture that comprises one or more amino acids selected from the groupconsisting of: L-isoleucine, L-tryptophan, and L-serine. In certainaspects, the one or more amino acids is added to the nutrient mixture ata concentration of about 1 mg/L, 10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L,300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, orabout 1000 mg/L. In certain aspects, the one or more amino acids isadded to the nutrient mixture at a concentration of about 400 mg/L.

Also presented herein are methods of enhancing TAG accumulation incultures of fungal microbes. In some aspects, the method can compriseadjusting the iron (Fe) content of a culture medium. In certain aspects,the method comprises increasing the iron concentration in the nutrientmixture by a factor of from two to as high as thirty, with a preferredconcentration of about 0.5 mM of iron per every 40 g/L of carbon source.

In some aspects, the method can comprise adding nickel to the nutrientmixture. In certain aspects, NiSO₄ is added in a concentration rangingfrom about 0.1 ppm, 1 ppm, 10 ppm, 100 ppm to about 1000 ppm. In certainaspects, NiSO₄ is added in a concentration ranging from about 1 ppm toabout 100 ppm of NiSO₄. In certain aspects, NiSO₄ is added in aconcentration ranging from about 1.1 ppm to about 11.1 ppm of NiSO₄.

In some aspects, the method can comprise adding malic acid to thenutrient mixture. In certain aspects, malic acid is added to thenutrient mixture before the culture is inoculated. In certain aspects,the malic acid concentration in the nutrient mix prior to inoculation isabout 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or about 10%.

In some aspects, the method can comprise adding to the nutrient mixtureone or more amino acids selected from the group consisting of:L-isoleucine, L-tryptophan, and L-serine. In certain aspects, the one ormore amino acids is added to the nutrient mixture at a concentration ofabout 1 mg/L, 10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L,500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, or about 1000 mg/L. Incertain aspects, the one or more amino acids is added to the nutrientmixture at a concentration of about 400 mg/L.

Also described herein is a biomass of a cellulose degrading fungus ofthe genus Penicillium, wherein the fungus comprises at least 5%triacylglyceride by weight. In certain aspects, the cellulose degradingfungus of the genus Penicillium comprises at least about 10% to about50% triacylglyceride by dry weight. In a preferred aspect, the cellulosedegrading fungus of the genus Penicillium comprises at least about 25%to about 50% triacylglyceride by dry weight.

In some aspects, the biomass has an enriched iron content. In someaspects, the biomass has an enriched malic acid content. In someaspects, the biomass has an enriched content of one or more amino acidsselected from the group consisting of: L-isoleucine, L-tryptophan, andL-serine.

In some embodiments described herein, compositions and methods areprovided which utilize fungal biomass for animal meal. In one aspect ofthe invention, an admixture is provided comprising fungal biomass andwhey. In some aspects, the fungal biomass comprises biomass derived froma cellulose degrading fungus of the genus Penicillium. In certainaspects, the admixture is suitable for animal and/or human consumption.

In some aspects, the biomass has been substantially depleted of a lipidproduct. In certain aspects, the lipid product comprisestriacylglycerides (TAG). In other aspects, the lipid product comprisesTAG and a phospholipid co-product. In certain aspects, the phospholipidco-product comprises lecithin.

In certain aspects, the biomass and the whey form a slurry. In certainaspects, the biomass and whey can be in a dry biomass/whey ratio rangingfrom about 1:100 to about 100:1 (vol/vol). In other aspects, the biomassand whey are in a dry biomass/whey ratio ranging from about 1:10 toabout 10:1 (vol/vol).

Also provided is a method of manufacture of an animal feed comprisingcombining dried fungal biomass with whey, thereby forming an admixture.In certain aspects, the method comprises, prior to combining: extractinga lipid product from fungal biomass; and, optionally, drying the fungalbiomass, thereby producing dried fungal biomass. In some aspects, thefungal biomass comprises biomass derived from a cellulose degradingfungus of the genus Penicillium. In certain aspects, the admixture issuitable for animal and/or human consumption.

In certain aspects, the lipid product extracted from the fungal biomasscomprises TAG. In other aspects, the lipid product comprises TAG and aphospholipid co-product. In certain aspects, the phospholipid co-productcomprises lecithin.

In certain aspects, the biomass and the whey form a slurry. In certainaspects, the biomass and whey can be in a dry biomass/whey ratio rangingfrom about 1:100 to about 100:1 (vol/vol). In other aspects, the biomassand whey are in a dry biomass/whey ratio ranging from about 1:10 toabout 10:1 (vol/vol).

Also presented herein is a method of shipping a fungal biomass-wheyslurry to a farm. In certain aspects the method comprises pumping wheyout of a truck. In certain aspects, the method additionally comprisespumping a fungal biomass-whey slurry into a tanker truck. In otheraspects, the slurry is pumped to a storage tank for animal feeding.

Also provided is a method of feeding an animal comprising providing tothe animal an admixture comprising fungal biomass and whey. In someaspects, the method further comprises identifying an animal that wouldlikely benefit from consuming the admixture. For example, identifying ananimal that would likely have a superior rate of growth or enhancedimmune system function if fed the fungal biomass or admixture describedherein as compared to if fed another feed. In some aspects, the fungalbiomass comprises biomass derived from a cellulose degrading fungus ofthe genus Penicillium. In certain aspects, the admixture is suitable foranimal and/or human consumption.

In some aspects, the biomass has been substantially depleted of a lipidproduct. In certain aspects, the lipid product comprises TAG. In otheraspects, the lipid product comprises TAG and a phospholipid co-product.In certain aspects, the phospholipid co-product comprises lecithin.

In certain aspects, the biomass and the whey form a slurry. In certainaspects, the biomass and whey can be in a dry biomass/whey ratio rangingfrom about 1:100 to about 100:1 (vol/vol). In other aspects, the biomassand whey are in a dry biomass/whey ratio ranging from about 1:10 toabout 10:1 (vol/vol).

Also presented is a method of feeding an animal comprising providing tothe animal a fungal biomass derived from a cellulose degrading fungus ofthe genus Penicillium.

Also presented herein is a method of improving the longevity of ananimal, comprising: identifying an animal in need of having improvedlongevity; and providing to the animal an animal feed comprising fungalbiomass derived from a cellulose degrading fungus of the genusPenicillium.

Also presented herein is a method of increasing the resistance of ananimal to an infection, comprising: identifying an animal in need ofincreased resistance to an infection; and proving to said animal ananimal feed comprising a fungal biomass derived from a cellulosedegrading fungus of the genus Penicillium.

With respect to any of the above-described methods, in certain aspects,the animal can be an aquatic animal. In certain other aspects, theanimal can be a terrestrial animal or avian. In certain aspects, thebiomass has been substantially depleted of a lipid product. In certainaspects, the lipid product comprises TAG. In other aspects, the lipidproduct comprises TAG and a phospholipid co-product. In certain aspects,the phospholipid co-product comprises lecithin.

In some aspects of the above above-described methods, the animal feedcan comprise the biomass in an amount ranging from 1% to 100% by weight.Accordingly, in some embodiments, the animal feed comprises fungalbiomass without whey. In some aspects, the animal feed can comprise thebiomass in an amount ranging from 5% to 10% by weight.

Also presented herein is an oxygenated mycelial mat comprising acellulose degrading fungus of the genus Penicillium. In certain aspects,the mycelial mat comprises dissolved oxygen in an amount greater than atleast 0.1 milligrams per liter.

Also presented herein is a mycelial mat comprising a cellulose degradingfungus of the genus Penicillium, the fungus comprising greater thanabout 25% (cdw) crude protein. In certain aspects, the fungus comprisesgreater than about 30% (cdw) crude protein.

Also presented herein is a mycelial mat comprising a cellulose degradingfungus of the genus Penicillium, said fungus comprising greater thanabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or greater than60% (cdw) TAG content.

With respect to any of the above-described compositions and/or methods,the cellulose degrading fungus of the genus Penicillium can be selectedfrom the group consisting of a fungus of the genus Penicillium that isthe same species as the fungus having NRRL deposit Accession No: 50410;a fungus comprising a 5.8 S ribosomal RNA gene sequence having at least98% nucleotide sequencing identity with the nucleic acid of SEQ ID NO:1; fungus comprising an ITS1 sequence with at least 98% sequenceidentity to SEQ ID NO: 2; a fungus comprising an ITS2 sequence with atleast 98% sequence identity to SEQ ID NO: 3; a fungus comprising a 28 Sribosomal RNA gene sequence with at least 98% sequence identity to SEQID NO: 4; and Penicillium menonorum.

Also presented herein is a conditioned medium comprising a substancethat inhibits the growth of an organism (hereinafter “inhibitorysubstance”), wherein the medium is conditioned by a cellulose degradingfungus. In certain aspects, the cellulose degrading fungus is a fungusof the genus Penicillium. In certain aspects, the inhibitory substanceis one or more compounds or other substances having an antibioticactivity. In some aspects, the inhibitory substance is one or morecompounds or other substances having an antimicrobial activity. Incertain aspects, the inhibitory substance is a bacteriostatic agent. Inother aspects, the inhibitory substance is a bacteriocidal agent.

Also presented herein is a process for producing an inhibitory substancecomprising: a) incubating a cellulose degrading fungus of the genusPenicillium in a liquid medium for a sufficient time for production ofan inhibitory substance by the fungus; and b) separating the fungus fromthe liquid medium, thereby producing a harvested fungus and aconditioned liquid medium, wherein the conditioned liquid mediumcomprises the inhibitory substance produced by the fungus. Accordingly,also presented herein is a conditioned medium containing an inhibitorysubstance, the conditioned medium produced by the process presentedherein. In certain aspects, the inhibitory substance is one or morecompounds or other substances having an antibiotic activity. In someaspects, the inhibitory substance is one or more compounds or othersubstances having an antimicrobial activity. In certain aspects, theinhibitory substance is a bacteriostatic agent. In other aspects, theinhibitory substance is a bacteriocidal agent.

The process for producing an inhibitory substance can further comprise:c) extracting the inhibitory substance from the conditioned medium.Accordingly, also presented herein is an extract comprising aninhibitory substance, the extract produced by the process presentedherein.

Also presented herein is a vessel comprising an inhibitory substanceobtained from a cellulose degrading fungus of the genus Penicillium. Incertain aspects, the inhibitory substance is one or more compounds orother substances having an antibiotic activity. In some aspects, theinhibitory substance is one or more compounds or other substances havingan antimicrobial activity. In certain aspects, the inhibitory substanceis a bacteriostatic agent. In other aspects, the inhibitory substance isa bacteriocidal agent.

Also presented herein is an extract of medium conditioned by a cellulosedegrading fungus of the genus Penicillium, said extract comprising oneor more extraction-soluble compounds having an activity inhibitory tothe growth and/or replication of one or more organisms. In some aspects,the compounds possess an antibiotic activity. In other aspects, thecompounds possess an antimicrobial activity. In certain aspects, theextract comprises one or more hexane soluble compounds. In certainaspects, the extract comprises one or more methy t-butyl ether solublecompounds. In certain aspects, the extract comprises one or moremethylene chloride soluble compounds. In certain aspects, the extractcomprises one or more ethyl acetate soluble compounds. In certainaspects, the extract comprises one or more amyl acetate solublecompounds. In certain aspects, the extract comprises one or moreisopropanol: methylene chloride (20%: 80%) soluble compounds. In certainaspects, the extract comprises one or more ethyl acetate (pH 2.5)soluble compounds. In certain aspects, the extract comprises one or moreamyl acetate (pH 2.5) soluble compounds.

With respect to any of the above-described compositions and/or methodsincluding conditioned medium, extracts thereof and processes forproducing the same, the cellulose degrading fungus of the genusPenicillium can be selected from the group consisting of a fungus of thegenus Penicillium that is the same species as the fungus having NRRLdeposit Accession No: 50410; a fungus comprising a 5.8 S ribosomal RNAgene sequence having at least 98% nucleotide sequencing identity withthe nucleic acid of SEQ ID NO: 1; fungus comprising an ITS1 sequencewith at least 98% sequence identity to SEQ ID NO: 2; a fungus comprisingan ITS2 sequence with at least 98% sequence identity to SEQ ID NO: 3; afungus comprising a 28 S ribosomal RNA gene sequence with at least 98%sequence identity to SEQ ID NO: 4; and Penicillium menonorum.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the claims and the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the present invention, both as to its structure andoperation, may be gleaned in part by study of the accompanying drawings,in which like reference numerals refer to like parts, and in which:

FIG. 1 is a flow chart of a cellulosic feedstock pretreatment processaccording to an embodiment of the invention.

FIG. 2 is a schematic of a bioreactor system for oxygenating fluidaccording to an embodiment of the invention.

FIG. 3 is a top view of the bioreactor system of FIG. 2.

FIG. 4 is a close up of fluid flow ramps, as shown in FIG. 2.

FIG. 5 is a flow chart of an inoculation and fermentation processaccording to an embodiment of the invention.

FIG. 6 is a flow chart of an inoculation and fermentation processaccording to an embodiment of the invention.

FIGS. 7A and 7B are block diagrams of a system for drying biomassaccording to an embodiment of the invention.

FIG. 8 is a block diagram of a system for drying biomass according to analternative embodiment of the invention.

FIG. 9 is a flow chart of a microbial biomass collection processaccording to an embodiment of the invention.

FIG. 10A is a flow chart of a TAG extraction and purification processaccording to an embodiment of the invention.

FIG. 10B is a flow chart of a TAG extraction and purification processaccording to another embodiment of the invention.

FIG. 11 is a flow chart of a biomass drying process according to anembodiment of the invention.

FIG. 12 is a flow chart of a separation process according to anembodiment of the invention.

FIG. 13 is a block diagram of process equipment used in accordance withFIGS. 1-12.

FIG. 14 depicts the sequence of the internal transcribed spacer (ITS) 1(SEQ ID NO: 2), 5.8 S rRNA gene (SEQ ID NO: 1), and ITS 2 (SEQ ID NO:3)and partial 28 S rRNA gene (SEQ ID NO: 4) nucleotide sequences. Theentire sequence shown in the figure is set forth as SEQ ID NO. 5, whichalso depicts the 225 nucleotides that are 5′ of ITS1.

FIG. 15 depicts a dendrogram of MM-P1, its closest known relatives, andother Penicillium (P.) and Eupenicillium (E.) strains, to showrelatedness of the species, as determined by parsimony analysis. Numbersabove the lines indicate bootstrap values for those nodes; onlybootstrap values over 70% are shown.

FIG. 16 depicts a Phylogenetic Tree of certain Penicillium (P.),Eupenicillium (E.), and Talaromyces (T.) species with occurrences ofsome important toxins noted.

FIG. 17 is a photograph of a growth-inhibition assay, and shows thatgrowth of Gram-positive bacterium Bacillus subtilis (light film) isinhibited (dark circles) by extracts from MM-P1 culture medium but notby a control sample of water.

FIG. 18 is an outline of the extraction procedure used in Example 10.

DETAILED DESCRIPTION OF THE INVENTION

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications. However, although variousembodiments of the present invention will be described herein, it isunderstood that these embodiments are presented by way of example only,and not limitation. As such, this detailed description of variousalternative embodiments should not be construed to limit the scope orbreadth of the present invention as set forth in the appended claims.

The described embodiments relate to systems and methods for productionof liquid biofuel from low-value starting materials. In someembodiments, the systems and methods relate specifically to theproduction of diesel, gasoline and/or aviation fuel from cellulosicfeedstocks. In some embodiments, the method includes a multi-stepprocess that inputs raw feedstock and outputs triacylglyceride (“TAG”)and aromatic compounds. In some embodiments, the systems and methodsrelate specifically to the production of biodiesel fuel. The productionof biodiesel fuel is further described in co-pending application Ser.No. 12/573,732, filed Oct. 5, 2009, entitled “Microbial Processing ofCellulosic Feedstocks for Fuel,” which is incorporated herein byreference.

Present methods of converting cellulosic biomass utilize biomassesspecifically cultivated for producing biofuels. In addition to these“cultivated cellulosic biomasses”, cellulosic biomass may be obtainedfrom cellulosic waste materials such as sawdust, wood chips, cellulose,algae, other biological materials, municipal solid waste (e.g., paper,cardboard, food waste, garden waste, etc.), and the like.

A process in accordance with an embodiment of the present inventionincludes converting cellulosic waste materials into liquid fuel. In oneaspect, cellulosic material such as agricultural waste is converted intolipids such as TAG, using specially selected or developed microbes(e.g., including genetically engineered microbes, competitively bredmicrobes, etc.). These microbes convert free sugars, cellulose andhemicellulose, major components of plant matter, into TAG.

TAG includes three fatty acids linked to a glycerol backbone. Whendissociated from the glycerol and hydrotreated, the fatty acids areconverted to hydrocarbons, which form the major components of diesel,gasoline and jet fuel. In some embodiments, TAG itself may serve as acomponent of fuel. A benefit associated with the present process is thatno net carbon is added to the atmosphere when the fuel is burned becausethe feedstock was originally produced by photosynthesis, sequesteringcarbon dioxide from the atmosphere.

Gasoline and jet fuel specifications require, in addition to alkanes, acertain proportion of aromatic compounds. TAG cannot be readilyconverted to aromatic compounds. However, plant matter also containslignin, a polymeric agglomeration of aromatic compounds that can bebroken down into the aromatics required for fuel. Specialized microbesattack lignin and convert it into smaller, individual aromaticcompounds. Thus, microbial conversion processes can suffice to convertagricultural and municipal waste originating from plant matter into allthe components of fuel.

Additionally, contemplated herein are compositions that include one ormore specific genera and species of microbes that not only convert awide range of cellulosic feedstocks into lipids effectively, but alsoyield desirable co-products. An example of the latter includes the spentmicrobial biomass after the lipids have been extracted, which can besold as protein meal to feed livestock in agriculture and aquaculture.The protein meal may also find use as a source of human nutrition.Metabolites produced and/or secreted by the one or more microbes provideanother class of co-products. In some embodiments, the metabolites maybe primary metabolites.

It will be appreciated that not every microbe is suitable for theconversion of cellulosic matter to lipids for fuel. For example, manymicrobes convert excess energy inputs into wax esters orpolyhydroxyalkanoates, which are less desirable than triaclyglycerides(TAG). Many microbes are pathogenic or otherwise unsuitable for animalor human consumption, or they provide insufficient nutritional value tomake a valuable nutritional co-product. Many microbes also do notproduce useful or economically valuable metabolite co-products.

In addition to the foregoing, the use of genetically modified organismsis discouraged because of concerns about environmental release. As such,specific genera and species of microbes that are suitable for efficientproduction of biofuels from cellulosic materials would be beneficial.Furthermore, specific genera and species of microbes that are capable ofand/or useful for both the production of biofuel and one or moreeconomically valuable co-products would be even more beneficial.

Described herein is a novel cellulose degrading fungus of the speciesPenicillium. It has been surprisingly found that this fungus can begrown in culture in vitro. It has also been surprisingly found that thisfungus can be grown in a bioprocess reactor under conditions that permitthe fungus to produce biofuel using a cellulosic carbon source.Furthermore, it has been surprisingly found that this fungus can begrown in a bioprocess reactor under conditions that permit the fungus toproduce biofuel and one or more economically valuable co-products.

The novel cellulose degrading fungus is designated herein as Penicilliummenonorum. In a preferred embodiment, the fungus comprises the straindesignated “MM-P1,” which is a wild-type strain of Penicillium menonorumthat was isolated from an environmental sample and surprisingly shown tohave the following desirable properties. First, Penicillium menonorumefficiently converts cellulosic sugars to lipids effectively. Second,Penicillium menonorum is able to store excess energy primarily in theform of TAG rather than or in addition to wax esters,polyhydroxyalkanoates or other substances. Third, Penicillium menonorumis non-pathogenic to animals and humans. Additionally, Penicilliummenonorum is a wild-strain microbe and has not been geneticallyengineered, thus alleviating concern over its release into theenvironment. In a preferred embodiment, the cellulose degrading funguscomprises a Penicillium species that is the same as the deposited strainhaving NRRL Accession No. 50410.

As used herein, the term “cellulose degrading” refers to the ability ofan organism to consume cellulose, hemicellulose, and/or othersaccharides derived from lignocellulosic materials. In some embodiments,cellulose degrading refers to an organism that produces enzymes thatdegrade cellulose and/or hemicellulose. In other embodiments, cellulosedegrading refers to an organism can break down cellulose and useportions of polymeric cellulose as an energy source.

In accordance with an embodiment of the present invention, a biomassfeedstock (e.g., sawdust, wood chips, cellulose, algae, other biologicalmaterials, or other solid materials) includes high-molecular-weight,high-energy-content molecules including cellulose, hemicellulose andlignin to be converted into fuel. The feedstock can generally bebiological matter, which generally includes organic compounds from plantor other lignocellulosic sources. The resulting fuel may be in fluidform, meaning that gaseous and liquid components may contribute to themake up of the fuel. For example, in one embodiment, the resulting fuelmay include methane (gas) and octane (liquid), as well as a variety ofother components. The feedstock material may be a low-value or wastematerial.

In certain embodiments of the present invention, a cellulosic biomassfeedstock includes at least 10% cellulosic waste materials. In someembodiments, the cellulosic biomass feedstock includes greater than 50%cellulosic waste materials. In still other embodiments, the cellulosicbiomass feedstock includes up to 100% cellulosic waste materials.

In one aspect, the feedstock may be a biological product of plantorigin, thus resulting in no net increase in atmospheric carbon dioxidewhen the resultant fuel product is combusted.

In some embodiments, two or more feedstocks may be used. For example, asecondary feedstock may include any material by-product of a celluloseconversion process, which material is capable of being converted intofuel by microbial action. The secondary feedstock may include glycerolmolecules or fragments thereof, or glycerol with additional carbon atomsor short paraffinic chains attached. Such compounds can be produced, forexample, when alkanes are cleaved from TAG or when TAG istransesterified to produce biodiesel.

For simplicity of explanation, a process in accordance with the presentinvention may be divided into three main steps: (1) feedstockpretreatment, (2) inoculation and fermentation/digestion, and (3)harvesting and extraction of the TAG and/or aromatic products.

(1) Feedstock Pretreatment

In an embodiment, raw feedstock is pretreated to make its carbon contentaccessible to microbial digestion and to kill any naturally presentmicrobes that might compete with the preferred species introduced forthe purpose of TAG and/or aromatic compound production. Pretreatment caninclude three steps: (1) mechanical pretreatment, (2) thermal-chemicalpretreatment and heat sterilization and/or ultraviolet (“UV”)irradiation and/or pasteurization, and (3) filtration/separation. In themechanical pretreatment step, raw feedstock may be conveyed to achopper, shredder, grinder or other mechanical processor to increase theratio of surface area to volume.

The thermal-chemical pretreatment step can treat the mechanicallyprocessed material with a combination of water, heat and pressure.Optionally, acidic or basic additives or enzymes may also be added priorto heat-pressure treatment. This treatment further opens up the solidcomponent (e.g., increases the ratio of surface area to volume) formicrobial access and releases sugars and other compounds (e.g.,dissociates cellulose and hemicellulose into component sugars, dimersand/or oligimers) into a liquid phase to make it more amenable tomicrobial digestion. Examples of such treatment include the class ofprocesses known variously as hydrolysis or saccharification, butlower-energy processing, such as simple soaking, boiling, or cooking inwater, may also be utilized.

In one embodiment, non-carbon microbial nutrients are added prior to thethermal-chemical pretreatment step. Non-carbon microbial nutrientsinclude, for example, sources of nitrogen, phosphorus, sulfur, metals,etc. After adding the non-carbon microbial nutrients, the entirety maythen be sterilized, such as via autoclaving. In some embodiments, thenon-carbon microbial nutrients are sterilized separately from the sugarcomponents. In some embodiments, the sugar components are notsterilized.

The filtration/separation step 140 preferably separates the solid matter(e.g., where the lignin is concentrated) from the liquid (e.g., whichcontains most of the sugars and polysaccharides from the cellulose andhemicellulose in the feedstock). In some embodiments, thefiltration/separation step 140 is optional. Consequently, in theseembodiments, the solid matter and liquid remain together throughoutprocessing.

In some embodiments, the feedstock is fortified (e.g., via the additionof glycerol.) For example, glycerol used in the feedstock fortificationmay be obtained as a byproduct of some TAG-to-alkanes conversionprocesses. Generally, glycerol is released by the conversion of TAG toproduce bio-diesel fuel (e.g. via transesterification). The releasedglycerol may then be metabolized to contribute to TAG formation. Abenefit of using the glycerol to form TAG is that it may speed thegrowth or TAG accumulation of certain microbial species duringfermentation, discussed below. It is understood that glycerol obtainedfrom transesterification is not high-purity, but rather includes avariety of constituents.

Referring now to FIG. 1, a flow chart of a cellulosic feedstockpretreatment process 100 in accordance with an embodiment of theinvention is shown. The pretreatment process 100 includes a receivingstage 110 for receiving the cellulosic feedstock and a mechanicalpretreatment stage 120 for transforming the feedstock into smallparticles.

The pretreatment process 100 also includes a thermo-chemicalpretreatment stage 130 to open up the cellulosic structure, renderingthe cellulosic structure more accessible to the microbes and to bringsome of the sugars and polysaccharides into solution. In someembodiments, water and, optionally, acidic or basic additives 134 areadded to the feedstock during this thermo-chemical pretreatment stage130. In some embodiments, non-carbon nutrients 138 used for themicrobial metabolization are also added during this thermo-chemicalpretreatment stage 130. In some embodiments, the non-carbon nutrients138 are sterilized separately from the feedstock in the thermal/chemicalpretreatment and sterilization step 130. Thereafter, the non-carbonnutrients 138 and the feedstock are combined after the solid/liquidseparation step 140. In other embodiments, the non-carbon nutrients andthe feedstock are combined prior to the solid/liquid separation step140. It should be appreciated that the thermo-chemical treatment step130 also serves to sterilize the cellulosic material and surroundingliquid to inhibit potentially competing microorganisms. In otherembodiments the non-carbon nutrients can be added to the liquid mediumafter the liquid/solid separation step.

The pretreatment process 100 also includes a solid-liquid separationstage 140 which may use mechanical means such as filters and/orcentrifuges to separate the bulk of the solid feedstock from the liquidportion. As described above, the liquid portion 144 includes mostlysugars and polysaccharides, while the solid portion 148 includes ligninas well as undissolved cellulose and hemicellulose.

(2) Inoculation and Fermentation

In the inoculation and fermentation stage, the solid and liquid portionsof the treated feedstock can be placed in separate digesters. In someembodiments the solid and liquid portions of the treated feedstock arenot placed in separate digesters. The digesters are vessels containingthe feedstock material and microbes that utilize the feedstock togenerate lipids and/or break down the feedstock into aromatic compounds,respectively, a solvent (e.g., water), and non-carbon nutrients (e.g.,nitrates, phosphates, trace metals, and the like).

The microbes may be species of any of two classes: one class whichconverts cellulose, hemicellulose or glycerol into lipids, and a secondclass which breaks lignin down into aromatic compounds. Microbesincluding bacterial and/or fungal species which convert cellulose,hemicellulose or glycerol into lipids include, for example, Trichodermareesi, Acinetobacter sp., and members of the Actinomyces andStreptomyces genera, some of which have been reported to store up to 80%of dry cell mass as lipids. In a preferred embodiment, the microbescomprise a cellulose degrading fungus of the genus Penicillium asdescribed in further detail below. Other species of bacteria and fungibreak lignin down into aromatics. In some embodiments, the microbes arefilamentatious or have a filamentatious morphology or structure. Thisfilamentatious morphology often results in chain growth of the microbes,which allows the microbes to be collected in traditional sieves orseparation means.

Isolated Fungus

In accordance with the above, described herein is a cellulose degradingfungus of the genus Penicillium. In some embodiments, the funguscomprises a fungus of the same species as the isolated Penicilliummenonorum strain MM-P1 deposited with the Agricultural Research ServiceCulture Collection (NRRL) on Aug. 2, 2010, having been assigned depositAccession No: 50410. The address of the depository, NRRL, is 1815 NorthUniversity Street, Peoria, Ill. 61604. This deposit has been made infull accordance with the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. In a preferred embodiment, the isolated fungus comprisesPenicillium menonorum and progeny thereof. In another preferredembodiment, the isolated fungus comprises NRRL deposit Accession No:50410 and progeny thereof.

In one embodiment, the isolated fungus can comprise a 5.8 S ribosomalRNA gene sequence as set forth in SEQ ID NO: 1. Accordingly, it will beappreciated that the isolated fungus can have a 5.8 S ribosomal RNA genesequence with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least99% nucleotide sequence identity with the nucleic acid of SEQ ID NO: 1.

In another embodiment, the isolated fungus can comprise an internaltranscribed spacer 1 (ITS1) sequence as set forth in SEQ ID NO: 2.Accordingly, it will be appreciated that the isolated fungus can have anITS1 sequence with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or atleast 99% nucleotide sequence identity with the nucleic acid of SEQ IDNO: 2.

In still another embodiment, the isolated fungus can comprise aninternal transcribed spacer 2 (ITS2) sequence as set forth in SEQ ID NO:3. Accordingly, it will be appreciated that the isolated fungus can havean ITS2 sequence with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% orat least 99% nucleotide sequence identity with the nucleic acid of SEQID NO: 3.

In yet another embodiment, the isolated fungus can comprise a 28 Sribosomal RNA gene sequence as set forth in SEQ ID NO: 4. Accordingly,it will be appreciated that the isolated fungus can have a 28 Sribosomal RNA gene sequence with at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or at least 99% nucleotide sequence identity with the nucleicacid of SEQ ID NO: 4.

In some embodiments, the isolated fungus having the above-describedsequence identity with SEQ ID NOs: 1, 2, 3 or 4 is of the genusPenicillium. As shown in FIGS. 15 and 16, the isolated fungus may havePenicillium striatisporum as its closest identified relative. In someembodiments, the isolated fungus is the teleomorph of Penicilliummenonorum. In some embodiments, the isolated fungus is the anamorph ofPenicillium menonorum.

A Nutrient Medium Comprising the Fungus (Fungal Cultures)

It will be appreciated the when an environmental sample ofmicroorganisms is obtained it cannot be predicted whether any of themicroorganisms can be extracted from the sample and cultivated in anisolated form. In fact, with many microorganisms, it cannot be predictedwhether the microbe can even be successfully cultured in vitro.

The microbes described herein can be grown and cultivated for storage orfor scale-up for use in the fermentation processes described elsewhereherein. Accordingly, presented herein is a living, in vitro culture of acellulose degrading fungus of the genus Penicillium growing on anutrient medium. In preferred embodiments, the fungus growing on thenutrient medium comprises Penicillium menonorum. Accordingly, in oneembodiment, the cellulose degrading fungus is of the genus Penicillium,the species being the same as NRRL deposit Accession No: 50410.

In certain embodiments, the medium is a solid medium. Techniques forpreparing solid nutrient growth media are well-known. For example, agelified nutrient medium such as an agar plate or dish may be used. Apreferred medium for growth of Penicillium menonorum is potato dextroseagar (PDA). Another preferred medium for growth of Penicillium menonorumis cornmeal agar. Another preferred medium for growth of Penicilliummenonorum is carboxymethylcellulose (CMC). It will be appreciated thatany other suitable solid medium, such as a grain, may be utilized. Thefungus may be transferred from the solid medium to other media and/orvessels by removing from a dish culture an agar plug having myceliumgrowing thereon and transferring the plug to the other media and/orvessels. In other embodiments, spores, such as conidiospores (conidia)can be transferred.

In some preferred embodiments, the nutrient medium can be a liquidmedium. Techniques for preparing solid nutrient liquid media arewell-known. Examples of nutrient-rich liquid culture media, such as maltextract broth and Sabouraud dextrose broth. Typically, liquid culturesare started by inoculating with isolated conidia or conidia obtainedfrom a plate culture.

Here, it has been surprisingly found that the novel Penicillium speciesPenicillium menonorum has enhanced growth and enhanced capacity toproduce molecules used in biofuel production and/or other co-products inboth liquid and solid medium when certain nutrients are present inspecified amounts and/or ratios. In some preferred embodiments, thegrowth and/or capacity to produce molecules used in biofuel productionand/or other co-products is optimized. Compositions comprising suchmedia provide conditions where the fungus can produce metabolic productsthat can be used in biofuel production and/or the production of othervaluable co-products. In a preferred embodiment, the media provideconditions where the fungus can produce such metabolic products in aneconomically feasible and/or economically efficient manner.

In a preferred embodiment, the fungal culture comprises a mediumcomprising a carbon source and a nitrogen source, wherein the ratio ofcarbon to nitrogen in the medium ranges from about 1:1 to about 1000:1.In a more preferred embodiment, the ratio of carbon to nitrogen in themedium ranges from about 20:1 to about 30:1, about 40:1, about 50:1,about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In a morepreferred embodiment, the ratio of carbon to nitrogen is 35:1 to about55:1 for a balance of biomass yield and TAG yield. Where biomass yieldis optimized, a ratio of about 20:1 to about 50:1, and preferably about23:1 is typical. In other preferred embodiments where TAG yield isoptimized, a larger ratio is typical, such as greater than about 100:1and preferably greater than 200:1.

The carbon source in the liquid or solid medium can be any suitablecarbon source for growth of Penicillium species in a liquid medium. Incertain embodiments, the carbon source can be one or more sugars ofnon-cellulosic origin. For example, the carbon source can comprise amixture of sugars such as fructose, glucose and sucrose. In anotherexemplary embodiment, the carbon source can be a juice material such ascane juice and/or its condensates, up to and including dry solidobtained by complete evaporation. Furthermore, the carbon source can befree sugars transported in a plant material, such as free-running sap ofplants. In another such aspect, the carbon source can comprise sugarsderived from corn starch.

In other preferred embodiments, the carbon source comprises a cellulosiccarbon source or a lysate thereof. For example, the cellulosic carbonsource can be derived from a biomass cultivated specifically forbiofuels production. Such cultivated cellulosic biomasses can includecrops such as algae, grasses, agricultural crops such as corn, soy,sorghum and the like. In addition to these “cultivated cellulosicbiomasses”, the cellulosic carbon source may be obtained from cellulosicwaste materials such as sawdust, wood chips, cellulose, algae, otherbiological materials, municipal solid waste (e.g., paper, cardboard,food waste, garden waste, etc.), and the like. In some preferredembodiments, the cellulosic carbon source is, or is a hydrolysate of,for example, an origin selected from the group consisting of sorghumgrain, other grains, stover of different grains; forage and othergrasses; oilseed crops and their stover; nut shells and hulls, includingalmond hulls; grape and other fruit pomace; yard and agricultural wasteof plant origin; algae; wood and wood byproducts including wood chips,bark and sawdust; paper products; animal manure especially including themanure of herbivorous animals; and food waste, especially food waste ofplant origin. However, it will be readily appreciated that any suitablecellulosic carbon source can be utilized with the cellulose degradingfungus described herein. In some embodiments the cellulosic carbonsource comprises other materials including, but not limited to,lignocelluloses and lysates thereof as well as hemicelluloses andlysates thereof.

As used herein with reference to cellulosic carbon sources, the term“lysate” and other like terms refer to a solubilized or partiallysolubilized cellulose structure. The term “hydrolysate” refers to asolubilized or partially solubilized cellulose structure produced by ahydrolysis process. Neither lysis nor hydrolysis should be understood torequire complete lysis of a cellulosic source material. Generation of alysate can involve loosening the cellulose structure and/or breakingdown or “clipping off” of cellulose chains to produce oligosaccharidesthat have a one or more beta 1,4 cellulosic linkage.

In some embodiments, the terms “cellulose” and “cellulosic” refer tomaterial comprising cellulose, and include for example, cellulose,hemicellulose, and lignocelluose.

Similarly, as used herein with reference to hemicellulose,hemicellulosic carbon sources, lignocellulose and lignocellulosic carbonsources, the term lysate and like terms refer to a solubilized orpartially solubilized cellulose structure derived from hemicellulose, ahemicellulosic material, lignocellulose or a lignocellulosic material.The term “hydrolysate” refers to a solubilized or partially solubilizedhemicellulosic or lignocellulose structure produced by a hydrolysisprocess. Neither lysis nor a hydrolysis should be understood to requirecomplete lysis of a hemicellulosic or lignocellulosic source material.For example, a lysate of lignocellulosic carbon source can containcellulose and hemicellulose chains that have been loosened fromtightly-bound lignin polymers. Generation of a lignocellulosic lysatecan involve loosening the cellulose structure and/or breaking down or“clipping off” of cellulose chains to produce oligosaccharides that havea one or more beta 1,4 cellulosic linkage.

In some embodiments, the medium preferably comprises at least onedissolved nutrient. Dissolved nutrients can be any carbon source,nitrogen source or any other mineral or nutrient source. For example,some complex nitrogen sources can include yeast extract, corn steep,mycelial cell extract from earlier cultures of the fungus, and the like.Other dissolved nutrients can include salts such as mineral salts,sulfates, nitrates, calcium salts. For example, the mineral salts caninclude ammonium salts including ammonium nitrate, ammonium sulfate, andthe like. Accordingly, the dissolved nutrients can include nutrientsselected from the group consisting of glycerol, yeast extract, cornsteep, mycelial cell extract from earlier cultures of the fungus,ammonium phosphate, magnesium sulfate, calcium chloride, ferric citrate,potassium sulfate, sodium acetate, sodium molybdate, copper sulfate,cobalt nitrate, zinc sulfate, boric acid and manganese chloride. It willbe appreciated that other nutrients may be selected and added as needed.

In some embodiments, the media can comprise MgSO₄ and/or other salt(s)of Mg at a concentration of at least 0.5 mM. Preferably, theconcentration of MgSO₄ and/or other salt(s) of Mg is at least about 0.5,1, 5, 10, 20, 30, 40 or at least 50 mM. Furthermore, it has beensurprisingly discovered that in the absence of a surfactant such asTween80, the addition of MgSO₄ and/or other salt(s) of Mg increases theamount of TAG produced by the culture when added at approximately 96hours after inoculation of a culture and increases resultant biomassyield when added at about time 0.

In certain embodiments, the nutrients and procedures described hereincan be modulated to significantly increase the accumulation of TAG byPenicillium menonorum and other candidate microbes, thus rendering theprocess more cost-effective. For example, it has been found thatstandard mineral nutrient recipes for culture of Penicillium do notnecessarily provide adequate iron for maximum cell growth and thusmaximum TAG accumulation by the population. A key enzyme in the lipidsynthesis pathway is Acetyl co-A synthetase. The active center of theenzyme, in some organisms, incorporates a nickel (Ni) ion [see, forexample, Ragsdale, S W (2009) Nickel-based Enzyme Systems. J. Biol.Chem. 284:18571-18575, hereby incorporated by reference in itsentirety]. The standard Penicillium culture nutrient formulationincludes no Ni. However, presented herein is the surprising discoverythat adding Ni to the culture medium of Penicillium menonorum canimprove TAG accumulation.

Additionally, provided herein is the surprising discovery that addingmalic acid to the culture medium of Penicillium menonorum can lead toincreased biomass and TAG accumulation.

Furthermore, provided herein is the surprising discovery that thepresence of tryptophan, serine and isoleucine can enhance P. menonorumgrowth and thus TAG accumulation by the population. In addition,tryptophan may specifically enhance TAG accumulation in each cell of thepopulation.

Accordingly, embodiments of the present disclosure comprise nutrientmixtures that provide for enhanced TAG accumulation in mixtures offungal microbes. In some aspects, the nutrient mixture comprisesincreased iron (Fe) content. In certain aspects, the nutrient mixturecomprises increased iron content wherein the iron content in thenutrient mixture is increased by a factor of about two to a factor ofabout thirty. In certain embodiments, the nutrient mixture comprises aconcentration of about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM,0.7 mM, 0.8 mM, 0.9 mM to about 1 mM of iron per every 40 g/L of carbonsource. In certain preferred embodiments, the nutrient mixture comprisesa concentration of about 0.5 mM of iron per every 40 g/L of carbonsource.

In some aspects, the nutrient mixture comprises Ni. In certain aspects,NiSO₄ is added in a concentration ranging from about 0.1 ppm, 1 ppm, 10ppm, 100 ppm to about 1000 ppm. In certain aspects, NiSO₄ is added in aconcentration ranging from about 1 ppm to about 100 ppm of NiSO₄. Incertain aspects, NiSO₄ is added in a concentration ranging from about1.1 ppm to about 11.1 ppm of NiSO₄.

In some aspects, the nutrient mixture comprises malic acid. In certainaspects, malic acid is added to the nutrient mixture before the cultureis inoculated. In certain aspects, the malic acid concentration in thenutrient mix prior to inoculation is about 0.01%, 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,or about 10%.

In some aspects, the nutrient mixture comprises one or more amino acidsselected from the group consisting of: L-isoleucine, L-tryptophan, andL-serine. In certain aspects, the one or more amino acids is added tothe nutrient mixture at a concentration of about 1 mg/L, 10 mg/L, 50mg/L, 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700mg/L, 800 mg/L, 900 mg/L, or about 1000 mg/L. In certain aspects, theone or more amino acids is added to the nutrient mixture at aconcentration of about 400 mg/L.

Also presented herein are methods of enhancing TAG accumulation incultures of fungal microbes. In some aspects, the method can compriseadjusting the iron (Fe) content of a culture medium. In certain aspects,the method comprises increasing the iron concentration in the nutrientmixture by a factor of from two to as high as thirty, with a preferredconcentration of about 0.5 mM of iron per every 40 g/L of carbon source.

In some aspects, the method can comprise adding nickel to the nutrientmixture. In certain aspects, NiSO₄ is added in a concentration rangingfrom about 0.1 ppm, 1 ppm, 10 ppm, 100 ppm to about 1000 ppm. In certainaspects, NiSO₄ is added in a concentration ranging from about 1 ppm toabout 100 ppm of NiSO₄. In certain aspects, NiSO₄ is added in aconcentration ranging from about 1.1 ppm to about 11.1 ppm of NiSO₄.

In some aspects, the method can comprise adding malic acid to thenutrient mixture. In certain aspects, malic acid is added to thenutrient mixture before the culture is inoculated. In certain aspects,the malic acid concentration in the nutrient mix prior to inoculation isabout 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or about 10%.

In some aspects, the method can comprise adding to the nutrient mixtureone or more amino acids selected from the group consisting of:L-isoleucine, L-tryptophan, and L-serine. In certain aspects, the one ormore amino acids is added to the nutrient mixture at a concentration ofabout 1 mg/L, 10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L,500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, or about 1000 mg/L. Incertain aspects, the one or more amino acids is added to the nutrientmixture at a concentration of about 400 mg/L.

In some embodiments, the medium comprises a non-ionic detergent such asPolysorbate 80 (Tween80), Polysorbate 20 (Tween20), deoxycholate,Brij-35 and the like. Preferably, the non-ionic detergent is Polysorbate80 (Tween80). More preferably, the liquid medium comprises Polysorbate80 (Tween80) at a concentration of at least 0.001%, 0.01% or at least0.1%. It has been surprisingly found that when MgSO₄ and/or othersalt(s) of Mg is added at about 96 hours in conjunction with Tween80added at time 0, both biomass and TAG yields are enhanced.

In preferred embodiments, the medium is at a pH of at about 2.5 to about10. More preferably, the pH is about 3.0 to about 7.0. More preferably,the pH is about 4.5 to about 6.5. More preferably, the pH is about 5.5.Maintaining a pH that is lower than neutral can help preventcontaminating growth of other organisms such as bacteria. In preferredembodiments, the temperature of the nutrient medium can range from about20° C. to about 50° C. More preferably, the temperature can range fromabout 28° C. to about 43° C. Where biomass and/or TAG yield is to beoptimized, the optimal temperature can range from about 36° C. to about39° C., with a preferred temperature of 37° C. It has been surprisinglydiscovered that the fungus grows well at a temperature of 37° C.

In embodiments where the fungus is cultivated in liquid culture, themedium preferably comprises dissolved oxygen in a range of about 0.01mg/L to about 1000 mg/L. Preferably, the dissolved oxygen is in a rangeof about 0.1 mg/L to about 100 mg/L. More preferably, the dissolvedoxygen is in a range of about 0.5 mg/L to about 40 mg/L.

In some embodiments, the microbes utilized in inoculation are grown instarter cultures using standard procedures. The standard procedures mayvary according to the particular species selected.

Fungal Culture in a Vessel

Additional embodiments described herein relate to a fungal culture in avessel, said culture comprising a liquid medium comprising a cellulosedegrading fungus of the genus Penicillium in growth phase, a carbonsource, a non-carbon nutrient mix, and MgSO₄ and/or other salt(s) of Mgat a concentration of at least 0.5 mM. In certain aspects, the cultureis approximately 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36,42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 120, 144, 168, 192, 216, 240 ormore than 240 hours old. More preferably, the culture is about 96 hoursold.

For growth of a culture in a vessel, the liquid medium can comprise acarbon source and a nitrogen source, wherein the ratio of carbon tonitrogen in the medium ranges from about 1:1 to about 1000:1. In a morepreferred embodiment, the ratio of carbon to nitrogen in the mediumranges from about 20:1 to about 30:1, about 40:1, about 50:1, about60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In a morepreferred embodiment, the ratio of carbon to nitrogen is 35:1 to about55:1 for a balance of biomass yield and TAG yield. Where biomass yieldis optimized, a ratio of about 20:1 to about 50:1, and preferably about23:1 is typical. In other preferred embodiments where TAG yield isoptimized, a larger ratio is typical, such as greater than about 100:1and preferably greater than 200:1.

The carbon source in the liquid medium can be any suitable carbon sourcefor growth of Penicillium species in a liquid medium. In certainembodiments, the carbon source can be one or more sugars ofnon-cellulosic origin. For example, the carbon source can comprise amixture of sugars such as fructose, glucose and sucrose. In anotherexemplary embodiment, the carbon source can be a juice material such ascane juice and/or its condensates, up to and including dry solidobtained by complete evaporation. Furthermore, the carbon source can befree sugars transported in a plant material, such as free-running sap ofplants. In another such aspect, the carbon source can comprise sugarsderived from corn starch.

In other preferred embodiments, the carbon source comprises a cellulosiccarbon source or a lysate thereof. For example, the cellulosic carbonsource can be derived from a biomass cultivated specifically forbiofuels production. Such cultivated cellulosic biomasses can includecrops such as algae, grasses, agricultural crops such as corn, soy,sorghum and the like. In addition to these “cultivated cellulosicbiomasses”, the cellulosic carbon source may be obtained from cellulosicwaste materials such as sawdust, wood chips, cellulose, algae, otherbiological materials, municipal solid waste (e.g., paper, cardboard,food waste, garden waste, etc.), and the like. In some preferredembodiments, the cellulosic carbon source is, or is a hydrolysate of,for example, an origin selected from the group consisting of sorghumgrain, other grains, stover of different grains; forage and othergrasses; oilseed crops and their stover; nut shells and hulls, includingalmond hulls; grape and other fruit pomace; yard and agricultural wasteof plant origin; algae; wood and wood byproducts including wood chips,bark and sawdust; paper products; animal manure especially including themanure of herbivorous animals; and food waste, especially food waste ofplant origin. However, it will be readily appreciated that any suitablecellulosic carbon source can be utilized with the cellulose degradingfungus described herein. In some embodiments the cellulosic carbonsource comprises other materials including, but not limited to,lignocelluloses and lysates thereof as well as hemicelluloses andlysates thereof.

The liquid medium preferably comprises at least one dissolved nutrientselected from the group consisting of glycerol, yeast extract, cornsteep, mycelial cell extract from earlier cultures of the fungus,sulfates, nitrates, calcium salts, ammonium phosphate, magnesiumsulfate, calcium chloride, ferric citrate, potassium sulfate, sodiumacetate, sodium molybdate, copper sulfate, cobalt nitrate, zinc sulfate,boric acid and manganese chloride. It will be appreciated that othernutrients may be selected and added as needed.

In some embodiments, the liquid medium comprises MgSO₄ and/or othersalt(s) of Mg at a concentration of at least 0.5 mM. Preferably, theconcentration of MgSO₄ and/or other salt(s) of Mg is at least about 0.5,1, 5, 10, 20, 30, 40 or at least 50 mM. It has been surprisinglydiscovered that in the absence of a surfactant such as Tween80, theaddition of MgSO₄ and/or other salt(s) of Mg increases the amount of TAGproduced by the culture when added at approximately 96 hours afterinoculation of a culture and increases resultant biomass yield whenadded at time 0.

In some embodiments, the liquid medium provides for enhanced TAGaccumulation in mixtures of fungal microbes. In some aspects, thenutrient mixture comprises increased iron (Fe) content. In certainaspects, the liquid medium comprises increased iron content wherein theiron content in the liquid medium is increased by a factor of about twoto a factor of about thirty. In certain embodiments, the liquid mediumcomprises a concentration of about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM to about 1 mM of iron per every 40g/L of carbon source. In certain preferred embodiments, the liquidmedium comprises a concentration of about 0.5 mM of iron per every 40g/L of carbon source.

In some aspects, the liquid medium comprises Ni. In certain aspects,NiSO₄ is added in a concentration ranging from about 0.1 ppm, 1 ppm, 10ppm, 100 ppm to about 1000 ppm. In certain aspects, NiSO₄ is added in aconcentration ranging from about 1 ppm to about 100 ppm of NiSO₄. Incertain aspects, NiSO₄ is added in a concentration ranging from about1.1 ppm to about 11.1 ppm of NiSO₄.

In some aspects, the liquid medium comprises malic acid. In certainaspects, malic acid is added to the liquid medium before the culture isinoculated. In certain aspects, the malic acid concentration in theliquid medium prior to inoculation is about 0.01%, 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,or about 10%.

In some aspects, the liquid medium comprises one or more amino acidsselected from the group consisting of: L-isoleucine, L-tryptophan, andL-serine. In certain aspects, the one or more amino acids is added tothe liquid medium at a concentration of about 1 mg/L, 10 mg/L, 50 mg/L,100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L,800 mg/L, 900 mg/L, or about 1000 mg/L. In certain aspects, the one ormore amino acids is added to the liquid medium at a concentration ofabout 400 mg/L.

In some embodiments, the liquid medium comprises a non-ionic detergentsuch as Polysorbate 80 (Tween80), Polysorbate 20 (Tween20),deoxycholate, Brij-35 and the like. Preferably, the non-ionic detergentis Polysorbate 80 (Tween80). More preferably, the liquid mediumcomprises Polysorbate 80 (Tween80) at a concentration of at least0.001%, 0.01% or at least 0.1%. It has been surprisingly found that whenMgSO₄ and/or other salt(s) of Mg is added at about 96 hours inconjunction with Tween80 added at about time 0, both biomass and TAGyields are enhanced.

In preferred embodiments, the liquid medium is at a pH of at about 3.0to about 7.0. More preferably, the pH is about 4.5 to about 6.5. Morepreferably, the pH is about 5.5. Maintaining a pH that is lower thanneutral can help prevent contaminating growth of other organisms such asbacteria. In preferred embodiments, the temperature of the nutrientmedium can range from about 20° C. to about 50° C. More preferably, thetemperature can range from about 28° C. to about 43° C. Where biomassand/or TAG yield is to be optimized, the optimal temperature can rangefrom about 36° C. to about 39° C., with a preferred temperature of 37°C.

The liquid medium preferably comprises dissolved oxygen in a range ofabout 0.01 mg/L to about 1000 mg/L. Preferably, the dissolved oxygen isin a range of about 0.1 mg/L to about 100 mg/L. More preferably, thedissolved oxygen is in a range of about 0.5 mg/L to about 40 mg/L.

In a preferred embodiment, the fungal culture in a vessel comprisesPenicillium menonorum. Preferably, the fungal culture in a vesselcomprises a fungus of the genus Penicillium, the species being the sameas NRRL deposit Accession No: 50410. In one preferred embodiment, thefungus can have a 5.8 S ribosomal RNA gene sequence with sequenceidentity with the nucleic acid of SEQ ID NO: 1. In another preferredembodiment, the fungus can have an ITS1 sequence having sequenceidentity with the nucleic acid of SEQ ID NO: 2. In still anotherpreferred embodiment, the fungus can have an ITS2 sequence havingsequence identity with the nucleic acid of SEQ ID NO: 3. In yet anotherpreferred embodiment, the fungus can have a 28 S rRNA gene sequencehaving sequence identity with the nucleic acid of SEQ ID NO: 4. Morepreferably, the sequence identity with SEQ ID NOs: 1, 2, 3 and/or 4 isat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%nucleotide sequence identity with the nucleic acid of SEQ ID NO: 1, 2, 3and/or 4.

The resultant lipids may include any molecular forms having astraight-chain saturated hydrocarbon portion. Such lipids are desirablebecause the straight-chain saturated hydrocarbon portion is relativelyeasy to convert to vehicle fuel.

Lipids include TAGs and wax esters. Mono- or di-unsaturated hydrocarbonchains are also found in lipids and are suitable for conversion toalkanes, albeit with the requirement of additional hydrogen to saturatethem.

The resultant aromatic compounds include any molecular forms havingcarbon ring structures. Examples of preferred aromatics include xylenes,methyl benzenes, and others.

In some embodiments, TAG and aromatic production is promoted bymaintaining the microbes in a high-carbon, low-nitrogen environment, andproviding aeration and/or agitation. As is understood, optimizing thepercentage of feedstock carbon converted to TAG or aromatics requirescontrolling the growth of the microbial culture so as to reduce thecarbon consumed by cell replication and metabolic activity and toincrease the carbon consumed in producing TAG and aromatics. This can bedone by controlling the ratio of non-carbon nutrient to carbon in thefeedstock, as well as by controlling other parameters such as pH,temperature, dissolved oxygen, carbon dioxide production, fluid shear,and the like. In some embodiments, one or more measurements of theseparameters may be used to determine when to harvest produced TAG. Forexample, in one embodiment, the carbon or nitrogen availability may bechanged in order to switch the culture from a rapid growth mode to a TAGaccumulation mode. In other words, one or more of these parameters mayhave a value associated with or which is indicative of desired TAGproduction.

In a preferred embodiment, nitrogen availability is controlled such thatwhen nitrogen is depleted, the production of TAG is stimulated. In someembodiments, nitrogen can be added during a culture run as needed tocontrol the production of TAG.

For example, in some embodiments, fluid shear is controlled by eithermoving the reactor vessel as a whole (e.g., by rocking it back and forthat a controlled frequency) or by means of mechanical agitators immersedin the fluid (e.g., any of a variety of paddle or stirrer shapes drivenby electrical motors at a controlled frequency).

In some embodiments, aeration or oxygenation of the fluid isaccomplished by any number of means, including via entrainment of airdue to turbulence caused by mechanical agitation of the fluid and viabubbling or sparging air, air enriched with oxygen, or pure oxygenthrough the fluid.

In some embodiments, oxygenation of the fluid is accomplished by usingturbulence to mix oxygen present in the head space of a bioreactor intothe liquid medium. As used herein, the head space is the space above theliquid medium that is occupied by gas and vapor. Without wishing to bebound by any particular theory, use of the head space for oxygenatingthe fluid is believed to result in a significantly reduced cost ofoperating an aerobic culture.

For example, referring to FIGS. 2-4, a bioreactor system 200 including abioreactor vessel 210 with head space 220 is shown. As presented,bioreactor vessel 210 is rotationally symmetric about a vertical axis(not shown). System 200 includes a rotary agitation mechanism 230 alongthe vertical axis that forces a strongly azimuthal flow when rotated (inaccordance with the arrow shown). For example, as shown, the rotaryagitation mechanism 230 includes a drive motor 232, a drive shaft 234,and a paddle 236.

As shown in FIG. 2, a rotary drive motor 232 and drive shaft 234 and alargely vertical paddle 236 induce a strongly azimuthal flow of theliquid medium about the center axis of the vessel 230. In oneembodiment, a flat paddle 236 with a radius equal to about one quarterof the bioreactor vessel 210's diameter and rotating at up to 150revolutions per minute is adequate to drive the desired flow. Theclearance between the outer radius of the paddle 236 and the inner edgeof the ramps 240 (discussed below) may be minimized, while avoidingimpact, rubbing or other physical contact between the two structures.

It will be appreciated that impeller and paddle design and the flowdirection are not restricted to those set forth above and in theFigures, but that any suitable type of impeller or flow direction can beused.

In an alternative embodiment, the agitation paddle 236 may be curved, soas to be concave in the direction of rotation. While not wishing to bebound by any particular theory, this change in paddle shape may generatea stronger azimuthal flow for a given paddle speed.

A set of stationary ramps 240 that force fluid to flow along a path withvariable vertical displacement are also shown within bioreactor vessel210. These ramps 240 may be secured to the vessel 210 via support rods212 and fasteners 214.

As shown in FIGS. 2 and 4, the topmost set of ramps 240 breaks theliquid surface 250 and is designed to cause a substantial fraction ofthe liquid to flow up the ramp 240 and fall back into the bulk liquid255. The turbulent re-entry of this “waterfall” entrains gases from thehead space 220 back into the bulk liquid 255. The lower-level ramps 240ensure that the gases thus entrained at the surface are well-mixed atall depths in the vessel 210.

In some embodiments, sets of ramps 240 may be used at various depths todeflect the fluid flow in the vertical direction to generate strongfluid mixing vertically. In such embodiments, the topmost set of ramps240 may extend across the mean liquid surface 250. It may be desirableto make sure that the topmost ramp's highest surface is low enough toallow most of the fluid to reach and flow over the top, falling freelyback into the liquid 255 and entraining gas from the head space 220,such as shown in FIG. 4.

In order to achieve improved results, ramps 240 having ramp angles of 30to 40 degrees, with the highest point of the topmost ramp 240 extendingapproximately 3 inches above the mean liquid surface 250 may be used. Insuch embodiments, the ramp's width is approximately one quarter of thebioreactor vessel 210's diameter, and two stacks of ramps 240 placeddiametrically opposite each other may be adequate to achieve the desiredmixing and oxygenation.

In one embodiment, ramps 240 having two horizontal end plates 240 a, band a flat, tilted plate section 240 c that generates the vertical floware used. The end plates 240 a, b may include slots 242 for ease ofmounting on vertical support rods 212 fixed in the bioreactor vessel 210(for example, by being fixed to a top cover plate). The slots 242 mayalso allow the ramp angle to be adjusted straightforwardly.

As shown in FIG. 2, two stacks 245 a, 245 b, of ramps 240 locateddiametrically opposite each other in the vessel 210 are shown. However,any number of stacks of ramps 240 (e.g., three, four, etc.) may be used.Also, as shown, there are two or three ramps 240 in each stack 245 a,245 b.

As described above, each ramp 240 includes three planar sections—twohorizontal end plates 240 a, 240 b and one tilted plate 240 c. The ramp240 can be made concave, or given raised lips along its inside andoutside edges, to form a trough that can send a larger proportion of theliquid over the lip of the waterfall, for increased gas entrainmentefficiency. Alternatively, the ramp 240 can be made as a single,continuous, curvilinear form along the azimuthal direction, rather thanout of three planar segments.

As shown in FIG. 4, the ramps 240 are rectangular in shape. In someembodiments, ramps 240 are made as circular arc segments to fit into thecircular bioreactor vessel 210, catch a larger fraction of the azimuthalflow and maintain a constant separation from the paddle 236.

Referring back to FIG. 2, support rods 212 may be vertical and threadedfor receiving threaded fasteners 214 to mount and hold the ramps 240.The ramps 240 could equally be attached to the side wall of thebioreactor vessel 210 via fastening means such as welding, adhesives, orthrough fasteners (e.g., bolts or rivets). In one embodiment, pair ofrods 212 is used to support each stack of ramps 240, however, any numberof rods 212 may be used.

As shown in FIG. 2, the rods 212 are suspended from a top plate (notshown) of the bioreactor vessel 210. However, rods 212 may alternativelyor also be anchored to the bottom of the bioreactor vessel 210. In someembodiments, the rods 212 are attached to a collapsible mechanism (notshown) that can be inserted through a smaller opening at the top of thevessel 210.

The bioreactor vessel 210 is shown as a flat-bottomed bioreactor.However, the design may be adapted to a conical-bottom bioreactor or adish-bottom reactor by reducing radii of ramps 240 and the agitationpaddle 236 appropriately, as a function of height in the conical ordished portion of the reactor.

Paddles 236, shafts 234, ramps 240, rods 212 and fasteners 214 may bemade of any material that (a) is strong enough to withstand peak andaverage loadings experienced in course of operation over time, (b) doesnot corrode in the aqueous culture with its range of pH and temperature,including sterilization temperatures, and (c) does not either interferewith the bioactivity of the culture or suffer biocorrosion. The defaultmaterial is stainless steel, but many different plastics and metals maybe used.

In an alternative embodiment, herein referred to as a “fountainreactor”, the reactor simply pumps the aqueous culture into a fountainin the head space, for example, via a spray fan, with the liquidentraining gases as it falls back into the main body of the liquid. Thepump can be a submersible, large-diameter pump to avoid clogging orfouling by the biomass generated by the culture. In some embodiments,the pump is mounted outside or external to the reactor.

Fungal Culture in a Bioprocess Reactor (Bioreactors)

In some embodiment, the bioreactor vessel described hereinabovecomprises a cellulose degrading fungus. For example, the bioreactorvessel may be a vessel as shown in FIGS. 2-4. However, it will bereadily appreciated that any suitable bioreactor system suitable for thegrowth of a cellulose degrading fungus can be used.

As used herein, the terms “bioprocess reactor”, “bioreactor vessel”,“bioreactor” and the like can refer to a vessel in which a biologicalprocess such as fermentation and microbial growth takes place.Bioprocess reactors such as those described herein and in the Figuresare useful for the growth of cellulose degrading fungi such as thefungus described herein as Penicillium menonorum. For example, in abioprocess reactor, any one of a number of variables such astemperature, pH, percent oxygenation, stir rate, and the like may bemonitored and/or controlled to achieve a desired growth condition.

Some bioreactor embodiments described herein include a bioprocessreactor comprising a cellulose degrading fungus of the genus Penicilliumgrowing in a liquid medium, wherein the fungus comprises at least 5%triacylglyceride by dry weight. In a preferred embodiment, the cellulosedegrading fungus of the genus Penicillium comprises at least about 10%to about 50% triacylglyceride by dry weight. In a more preferredembodiment, the cellulose degrading fungus of the genus Penicilliumcomprises at least about 25% to about 50% triacylglyceride by dryweight. In a preferred embodiment, the fungus comprises Penicilliummenonorum.

In preferred embodiments, the bioprocess reactor comprises a fungus ofthe genus Penicillium growing in a liquid medium, said liquid mediumcomprising a carbon source and a nitrogen source, wherein the ratio ofcarbon to nitrogen in the medium ranges from about 1:1 to about 1000:1.

In another embodiment, the bioprocess reactor comprises a cellulosiccarbon source or a lysate thereof having a cellulose degrading fungus ofthe genus Penicillium growing thereon. In a preferred embodiment, thecellulosic carbon source is present in a liquid medium.

In another embodiment, the bioprocess reactor comprises a cellulosedegrading fungus of the genus Penicillium growing in a liquid medium,wherein the liquid medium comprises at least one lipid selected from thegroup consisting of triacylglycerides and phospholipids. In someembodiments, the liquid medium further comprises glycerol.

It will be readily understood that the terms glycerol, glycerin andglycerine and like terms refer to the same organic compound, also knownas propan-1,2,3-triol or 1,2,3-trihydroxypropane.

For growth of a culture in a bioprocess reactor, the liquid medium cancomprise a carbon source and a nitrogen source, wherein the ratio ofcarbon to nitrogen in the medium ranges from about 1:1 to about 1000:1.In a more preferred embodiment, the ratio of carbon to nitrogen in themedium ranges from about 20:1 to about 30:1, about 40:1, about 50:1,about 60:1, about 70:1, about 80:1, about 90:1 or about 100:1. In a morepreferred embodiment, the ratio of carbon to nitrogen is 35:1 to about55:1 for a balance of biomass yield and TAG yield. Where biomass yieldis optimized, a ratio of about 20:1 to about 50:1, and preferably about23:1 is typical. In other preferred embodiments where TAG yield isoptimized, a larger ratio is typical, such as greater than about 100:1and preferably greater than 200:1.

The carbon source in the liquid medium in the bioprocess reactor can beany suitable carbon source for growth of Penicillium species in a liquidmedium. In certain embodiments, the carbon source can be one or moresugars of non-cellulosic origin. For example, the carbon source cancomprise a mixture of sugars such as fructose, glucose and sucrose. Inanother exemplary embodiment, the carbon source can be a juice materialsuch as cane juice and/or its condensates, up to and including dry solidobtained by complete evaporation. Furthermore, the carbon source can befree sugars transported in a plant material, such as free-running sap ofplants. In another such aspect, the carbon source can comprise sugarsderived from corn starch.

In other preferred embodiments, the carbon source comprises a cellulosiccarbon source or a lysate thereof. For example, the cellulosic carbonsource can be derived from a biomass cultivated specifically forbiofuels production. Such cultivated cellulosic biomasses can includecrops such as algae, grasses, agricultural crops such as corn, soy,sorghum and the like. In addition to these “cultivated cellulosicbiomasses”, the cellulosic carbon source may be obtained from cellulosicwaste materials such as sawdust, wood chips, cellulose, algae, otherbiological materials, municipal solid waste (e.g., paper, cardboard,food waste, garden waste, etc.), and the like. In some preferredembodiments, the cellulosic carbon source is, or is a hydrolysate of,for example, an origin selected from the group consisting of sorghumgrain, other grains, stover of different grains; forage and othergrasses; oilseed crops and their stover; nut shells and hulls, includingalmond hulls; grape and other fruit pomace; yard and agricultural wasteof plant origin; algae; wood and wood byproducts including wood chips,bark and sawdust; paper products; animal manure especially including themanure of herbivorous animals; and food waste, especially food waste ofplant origin. However, it will be readily appreciated that any suitablecellulosic carbon source can be utilized with the cellulose degradingfungus described herein. In some embodiments the cellulosic carbonsource comprises other materials including, but not limited to,lignocelluloses and lysates thereof as well as hemicelluloses andlysates thereof.

In some embodiments, the liquid medium in the bioprocess reactorpreferably comprises at least one dissolved nutrient selected from thegroup consisting of glycerol, yeast extract, corn steep, mycelial cellextract from earlier cultures of the fungus, sulfates, nitrates, calciumsalts, ammonium phosphate, magnesium sulfate, calcium chloride, ferriccitrate, potassium sulfate, sodium acetate, sodium molybdate, coppersulfate, cobalt nitrate, zinc sulfate, boric acid and manganesechloride. It will be appreciated that other nutrients may be selectedand added as needed.

In some embodiments, the liquid medium in the bioprocess reactorcomprises MgSO₄ and/or other salt(s) of Mg at a concentration of atleast 0.5 mM. Preferably, the concentration of MgSO₄ and/or othersalt(s) of Mg is at least about 0.5, 1, 5, 10, 20, 30, 40 or at least 50mM. It has been surprisingly discovered that in the absence of asurfactant such as Tween80, the addition of MgSO₄ and/or other salt(s)of Mg increases the amount of TAG produced by the culture when added atapproximately 96 hours after inoculation of a culture and increasesresultant biomass yield when added at about time 0.

In some embodiments, the liquid medium in the bioprocess reactorprovides for enhanced TAG accumulation in mixtures of fungal microbes.In some aspects, the nutrient mixture in the bioprocess reactorcomprises increased iron (Fe) content. In certain aspects, the liquidmedium in the bioprocess reactor comprises increased iron contentwherein the iron content in the liquid medium is increased by a factorof about two to a factor of about thirty. In certain embodiments, theliquid medium in the bioprocess reactor comprises a concentration ofabout 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM,0.9 mM to about 1 mM of iron per every 40 g/L of carbon source. Incertain preferred embodiments, the liquid medium in the bioprocessreactor comprises a concentration of about 0.5 mM of iron per every 40g/L of carbon source.

In some aspects, the liquid medium in the bioprocess reactor comprisesNi. In certain aspects, NiSO₄ is added in a concentration ranging fromabout 0.1 ppm, 1 ppm, 10 ppm, 100 ppm to about 1000 ppm. In certainaspects, NiSO₄ is added in a concentration ranging from about 1 ppm toabout 100 ppm of NiSO₄. In certain aspects, NiSO₄ is added in aconcentration ranging from about 1.1 ppm to about 11.1 ppm of NiSO₄.

In some aspects, the liquid medium in the bioprocess reactor comprisesmalic acid. In certain aspects, malic acid is added to the liquid mediumbefore the culture is inoculated. In certain aspects, the malic acidconcentration in the liquid medium prior to inoculation is about 0.01%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, or about 10%.

In some aspects, the liquid medium in the bioprocess reactor comprisesone or more amino acids selected from the group consisting of:L-isoleucine, L-tryptophan, and L-serine. In certain aspects, the one ormore amino acids is added to the liquid medium at a concentration ofabout 1 mg/L, 10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L,500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, or about 1000 mg/L. Incertain aspects, the one or more amino acids is added to the liquidmedium at a concentration of about 400 mg/L.

In some embodiments, the liquid medium in the bioprocess reactorcomprises a non-ionic detergent such as Polysorbate 80 (Tween80),Polysorbate 20 (Tween20), deoxycholate, Brij-35 and the like.Preferably, the non-ionic detergent is Polysorbate 80 (Tween80). Morepreferably, the liquid medium comprises Polysorbate 80 (Tween80) at aconcentration of at least 0.001%, 0.01% or at least 0.1%. It has beensurprisingly found that when MgSO₄ and/or other salt(s) of Mg is addedat about 96 hours in conjunction with Tween80 added at about time 0,both biomass and TAG yields are enhanced.

In preferred embodiments, the liquid medium in the bioprocess reactor isat a pH of at about 2.5 to about 10. More preferably, the pH is about3.0 to about 7.0. More preferably, the pH is about 4.5 to about 6.5.More preferably, the pH is about 5.5. Maintaining a pH that is lowerthan neutral can help prevent contaminating growth of other organismssuch as bacteria. In preferred embodiments, the temperature of thenutrient medium can range from about 20° C. to about 50° C. Morepreferably, the temperature can range from about 28° C. to about 43° C.Where biomass and/or TAG yield is to be optimized, the optimaltemperature can range from about 36° C. to about 39° C., with apreferred temperature of 37° C. It has been surprisingly discovered thatthe fungus grows well at a temperature of 37° C.

In certain preferred embodiments, the medium in the bioprocess reactorpreferably comprises dissolved oxygen in a range of about 0.01 mg/L toabout 1000 mg/L. Preferably, the dissolved oxygen is in a range of about0.1 mg/L to about 100 mg/L. More preferably, the dissolved oxygen is ina range of about 0.5 mg/L to about 40 mg/L.

In another preferred embodiment, the bioprocess reactor comprisesPenicillium menonorum. Preferably, the fungal culture comprises a fungusof the genus Penicillium, the species being the same as NRRL depositAccession No: 50410.

In one preferred embodiment, the fungus in the bioprocess reactor canhave a 5.8 S ribosomal RNA gene sequence with sequence identity with thenucleic acid of SEQ ID NO: 1. In a preferred embodiment, the fungus canhave an ITS1 sequence having sequence identity with the nucleic acid ofSEQ ID NO: 2. In a preferred embodiment, the fungus can have an ITS2sequence having sequence identity with the nucleic acid of SEQ ID NO: 3:In a preferred embodiment, the fungus can have a 28 S rRNA gene sequencehaving sequence identity with the nucleic acid of SEQ ID NO: 4. Morepreferably, the sequence identity with SEQ ID NOs: 1, 2, 3 and/or 4 isat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%nucleotide sequence identity with the nucleic acid of SEQ ID NO: 1, 2, 3and/or 4.

In some embodiments of the bioprocess reactors described herein, thebioprocess reactor further comprises an impeller rotating at a stir rateof about 1 RPM to about 1200 RPM. In some embodiments, the stir rate isabout 1 RPM to about 300 RPM. In certain aspects the stir rate is about20 to about 100 RPM. In certain aspects, the stir rate is about 60-70RPM.

An example illustrating benefits of the bioreactor system 200 will nowbe described.

EXAMPLE

Experiments were conducted on two 100-liter microbial cultures that wereidentical except for the oxygenation mechanism. The first culture (e.g.,control sample) used 10 liters per minute of oxygen-enriched air, pumpedthrough a perforated-tube sparger at the bottom of the bioreactor. Thesecond culture (e.g., experimental sample) used the “waterfall” methodexemplified by bioreactor system 200 and pumped the same 10 liters perminute of identically enriched air into the headspace.

On the fourth day of the culture, at a time of maximal growth (and,hence, maximal oxygen consumption), the dissolved oxygen in the spargedreactor (e.g., control sample) measured 8 to 9 milligrams of O₂ perliter of liquid, while the dissolved oxygen in the waterfall reactor(e.g., experimental sample) measured 17 to 18 milligrams per liter.Based on this example, the waterfall reactor produced a higher dissolvedoxygen content (by 8-10 milligrams per liter) than a traditional spargedreactor.

In accordance with some embodiments, using the waterfall method includesoptionally, enriching the gas in the head space of an aqueous bioreactorby pumping in air enriched with oxygen by a commercially availableoxygen enricher. Because the enriched gas is being pumped into a largemanifold, there is no requirement of high pressure pumping, in contrastto approaches relying on bubbling (sparging).

Benefits of the using the bioreactor vessel 210 and the waterfall methodinclude:

-   -   No high output pressure oxygen enrichment required;    -   No high-pressure gas flow required;    -   No perforated or sintered sparging apparatus to suffer        biofouling;    -   No moving parts other than the rotary agitator;    -   Very low cost;    -   Highly effective oxygenation of the culture, even under heavy        growth conditions that are often susceptible to oxygen        deprivation.

Referring now to FIG. 5, a flow chart of an inoculation and fermentationprocess 500 in accordance with an embodiment of the invention is shown.The inoculation and fermentation process 500 includes a receiving stage510 for receiving the liquid output 144 from the pretreatment process100 and an inoculation step 520 that adds a starter culture 525 of theselected microorganism or microorganisms to the liquid 144 to form amixture at the inoculation step 520. The selected microorganisms may bea single species or strain, or a combination of multiple species orstrains. In a preferred embodiment, the microorganism is Penicilliummenonorum. The inoculum can be in the form of vegetative cells orspores.

The inoculation and fermentation process 500 also includes ametabolization step 530, which takes the mixture and controls parameterssuch as temperature, pH, dissolved oxygen, and fluid shear usingappropriate methods known in the art. During this metabolization step530, the microorganisms proliferate and metabolize the feedstock,creating intracellular inclusions of lipids in the form oftriacylglycerides (TAG). At the end of this stage (e.g., as determinedby defined values of one or more of the parameters of time, pH,dissolved oxygen or others) the metabolization is stopped, yielding adepleted fluid 540 with suspended microbes containing TAG.

Referring now to FIG. 6, a flow chart of an inoculation and fermentationprocess 600 in accordance with an embodiment of the invention is shown.The inoculation and fermentation process 600 includes a receiving stage610 for receiving the solid portion of the pretreated feedstock 148 fromthe pretreatment process 100 and an inoculation step 620, in which theportion of feedstock 148 is mixed with sterilized water and non-carbonnutrients 624 and a starter culture of specially selected microorganismssuited to decomposing the lignin 628. The selected microorganisms may bea single species or strain, or a combination of multiple species orstrains. In a preferred embodiment, the microorganism is Penicilliummenonorum. The inoculum can be in the form of vegetative cells orspores.

The inoculation and fermentation process 600 also includes ametabolization step 630, which takes this mixture and controlsparameters such as temperature, pH, dissolved oxygen, and fluid shearusing appropriate methods known in the art. During this metabolizationstep 630, the microorganisms proliferate and metabolize the feedstock,breaking the lignin down into smaller aromatic compounds that arereleased into the solution. At the end of this stage (e.g., asdetermined by defined values of one or more of the parameters of time,pH, dissolved oxygen or others) the metabolization is stopped, yieldinga mixture 640 containing depleted solids, microbes, and gas and liquidcontaining the desired aromatic compounds.

(3) Harvesting and Product Extraction

Extracting product from a fermentor is different, depending on whetherthe product is TAG or protein-rich fungal biomass. Each is considered inturn. In both cases, however, choosing the proper time to harvest willmaximize yield. Measurements such as pH, dissolved oxygen, carbondioxide production, remaining carbon nutrient concentration, and thelike can be used to determine the optimal harvest time.

In particular, it has been surprisingly found that during the growthphase (also called “log phase” and “linear phase”) of the culture, whenmicrobial cells are dividing rapidly, protein content of the cells ishigh (approximately 32.2% of cell dry weight, (cdw)) and TAG content islow (typically approximately <6% of cdw). When the nitrogen in theculture medium is low or exhausted but sufficient carbohydrates stillexist in the medium, the log phase ceases and a nutritional stress phasecommences, where the cells accumulate TAG. In this regime, not only doesTAG increase as a percentage of cdw, but the protein content decreases.In fact, the protein content decreases not only as a fraction of totalcdw, but also as a fraction of the dry weight of cells after the TAG hasbeen extracted (residual cdw). Accordingly, the ideal time to harvestdepends on whether fuel or animal feed is the primary market drivingforce, and on what amount of protein as a percentage of cdw is desiredfor the animal feed, and harvest time can be adjusted as market demandschange. If fuel is the main desired product, harvest can be delayeduntil TAG forms >30% of cdw; then the protein content may be as low as<15% of residual cdw. If protein-rich feed is the primary product,harvest can be performed right at the end of the growth phase, when TAGforms <6% of cdw and protein content may be as high as 25% of residualcdw, or higher. If a lower protein content feed is desired, harvest canbe performed after the end of the growth phase but before the amount ofTAG reaches its maximum percentage of cdw.

Harvesting and Extracting TAG

The liquid medium in the digesters has provided TAG-producing microbeswith nourishment, allowing the microbes to flourish and reproduce. Thesemicrobes store TAG in intracellular structures. The first step,accordingly, is to harvest or collect the cellular biomass from theliquid medium. Because cells tend to form multicellular agglomerationshundreds of micrometers in size, harvesting may be performed byscreening, sieving, centrifugation, or filtration. The multicellularagglomerations may be the result of the filamentatious nature of themicrobes, as discussed above. The result of this step is a mass ofcellular biomass with remaining excess water, e.g. wet biomass.

In some embodiments, the wet biomass is dried after the collecting step.For example, gross excess water may be removed mechanically from the wetbiomass by pressing through a roller press, squeezed, or wrung.Alternatively, water may be removed using a fan separator, screw press,or by spray harvesting techniques. The wet biomass may then be furtherdried using a vacuum oven, lyophilizer, or other common dryingequipment. It should be recognized that when using a vacuum oven, forexample, the temperature should be controlled so that TAG is not or isonly minimally hydrolyzed (e.g., drying at temperatures of about lessthan 80° C.). In some embodiments, lyophilizing is selected as thedrying means because it has the effect of increasing the surface area tovolume ratio of the biomass, which makes subsequent extraction moreefficient. In some embodiments, flash freezing (e.g., via immersion inliquid nitrogen or −80° C. freezer) is used to break up the cellstructures, improving efficiency of subsequent extraction, and possiblypartially disrupting the cells.

Because the wet biomass may be degraded when the water is removed,drying the biomass both efficiently and gently is preferred. In order toachieve gentle drying, processes in accordance with an embodimentgenerally do not expose or minimize exposure of the biomass to heat.

Additionally, when drying biomass, it may be important to avoidreleasing live microbes or spores into the environment. Consequently,microbial cultivators and processors often ensure that any biologicalmaterial is sterilized prior to release in the atmosphere, drain ortrash. In an embodiment, the described apparatus and method ensurebiological material is sterilized, thereby avoiding releasing livecontaminants (e.g., microbes or spores) into the environment.

In some embodiments, a method of drying the biomass includes exposingthe biomass to low-humidity air or dry, inert gas and then sterilizingthe air, thereby killing any microbes or spores or other biologicalmaterial entrained in the air.

An exemplary system for drying the biomass is provided in FIG. 7A. Asshown in FIG. 7A, system 700 includes a dehumidifying chamber 702, adrying chamber 705, and a sterilization chamber 707. System 700 mayadditionally include a fluid intake section 701, a fluid pump 703, andhoses or connectors 704. Drying chamber 705 may include one or moreperforated drying screens 706. Sterilization chamber 707 may include asterilization or biocide fluid 708. As used herein, “fluid” includesgases such as air, as well as liquids.

In one embodiment, the system 700 first provides low-humidity air or dryinert gas via fluid intake section 701 and dehumidifying chamber 702.Then, the dry inert gas or low-humidity air is moved past the biomass tobe dried in drying chamber 705. The air or inert gas is thereaftersterilized in sterilization chamber 707, thereby killing any microbes orspores or other biological matter entrained in the air. The arrowsindicate air flow.

As is appreciated, the source of low-humidity air may be a dehumidifier.Alternatively, when a dry, inert gas is used, the source may be acompressed gas cylinder filled with a suitable gas such as nitrogen orhelium.

Drying chamber 705 may be any container such as a metal or plastic boxwith sufficient volume to contain the material (e.g., biomass) to bedried. Drying chamber 705 typically includes an input duct or portcoupled to the source of low-humidity air (e.g., dehumidifying chamber702), and an output duct or port coupled to the sterilization chamber707. The material to be dried may be arranged or spread thinly upon onone or more shelves or platforms (e.g., perforated drying screens 706)supported within the drying chamber 705, preferably arranged so that theair passes freely over the material. The shelves may be porous orgrid-like to permit evaporation from both top and bottom surfaces of thematerial.

Referring to FIG. 7B, in some embodiments, perforated drying screens 706have a mesh screen 710 placed on top of the drying screens 706.Thereafter, the biomass may be added to the drying chamber 705 bypumping the biomass into the top of the drying chamber 705 frombioreactor 200 via tubing and/or pump 712. Air may be bubbled up frombeneath the drying screens 706 from an air source 713, where eachperforation 706 a may have a microchannel 706 b attaching it to the airsource 713 (e.g., a bubbler). In one embodiment, the air source 713 isthe same air source as used in FIG. 7A. The air flowing through thescreens 706 helps the liquid to flow out of the biomass and through themesh 710. Additionally, in some embodiments, drying chamber 705 includesa stirrer 722 and/or a heater 724 in the space above mesh 710. While notwishing to be bound by any particular theory, it is believed thatstirrer 722 assists the liquid flowing through mesh 710, while trappingthe biomass on mesh 710, and heater 724 provides drying to the biomass.Any resulting liquid may be pumped out of the bottom of the tank 705 viatubing and/or pump 714 and recycled back into bioreactor 200. In someembodiments, a dehumidifier and various types of heat can also be used(e.g., heat the air, heat the tank, etc.).

Upon reaching a sufficient dryness, the biomass can be removed fromdrying chamber 705 via traditional mechanical or pneumatic means and/orautomated means. For example, suitable removal means include, but arenot limited to, scoops, conveyors, valves, and compressed air or vacuum.

Additionally, while not explicitly shown, the drying chamber maycomprise a tumble-dry configuration whereby the material to be dried isagitated with the low-humidity air. Alternatively, any other dryingconfiguration which results in a moisture content of less than or equalto about 10% may be used.

Sterilization chamber 707 may be any container such as a metal orplastic box with sufficient volume to contain the means for sterilizingthe air and any entrained biological matter. Sterilization chamber 707typically includes an input port or duct coupled to the exit port of thedrying chamber 705, and an output port or duct through which sterilizedair is released. The output port may include a filter (not shown) tofurther contain biological material.

The sterilizing means may be any means for killing microbes, spores, orother biological matter entrained in the passing air, prior to release.The sterilizing means may be a drum or bucket containing water andbleach through which the air is bubbled. Alternatively, the sterilizingmeans may be ultraviolet lamps producing radiation with sufficientintensity and wavelength to kill cells in the passing air.

As provided above, system 700 may additionally include a fluid pump 703.Fluid pump 703 may be any type of blowing apparatus such as a fan orpump. Fluid pump 703 preferably provides sufficient airflow volume toeffectively remove moisture from the material to be dried, andsufficient pressure to pass the air through the chambers 702, 705, 707,including the sterilization chamber 707 back-pressure if present. Insome embodiments, fluid pump 703 includes multiple fans or blowers. Forexample, dehumidifying chamber 702 may include a built-in fan to urgeair into the drying chamber 705, and another fan may be positionedbetween the drying chamber 705 to urge air into the sterilizationchamber 707 and a third fan may be arranged to draw air out of thesterilization chamber 707 and pass the air through a filter beforerelease.

The system 700 may be open-cycle or closed-cycle. An open-cycle systemdraws air from the environment or other source such as a compressed-gascylinder, dehumidifies it if necessary, passes it over the material tobe dried, sterilizes it, and then releases it to the environment. Aclosed-cycle system passes the air from the sterilization chamber 707back to the dehumidifying chamber 702 for re-use. The open-cycle systemis simpler, but the closed-cycle system may be more economical whenusing an alternative gas such as nitrogen for drying.

In an exemplary embodiment, to remove water from the wet biomass, thebiomass is spread in a thin layer across the screens 706. Perforationsin the screens allow the air or dry gas access to both upper and lowersurfaces of the biomass layer. The chamber 705 is then closed.

An air dehumidifier or cylinder of compressed dry gas (e.g., nitrogen orhelium—any gas that is inert with respect to biological materials) isconnected to a fan or pump 703 to force it through the drying chamber705, largely parallel to the layers. The fan or pump 703 may beconnected at the inlet, forcing the air through; or it may be connectedat the outlet, using a vacuum to draw the air through; or both. The drygas or dehumidified air flowing past the layer effectively draws waterfrom the biomass.

During this process, the gas may pick up microscopic amounts ofbiological material, including cells of the microbial culture, as itpasses through the drying chamber 705. To prevent spreading ofpotentially undesirable organisms, in one embodiment, the air is bubbledthrough a reservoir of bleach or other biocidal liquid upon exiting thedrying chamber 705 in sterilization chamber 707 before being releasedinto the atmosphere. Alternatively, the air may pass through a region ofintense ultraviolet illumination that also acts as a biocide. Thisalternative embodiment is shown in FIG. 8, described below.

Referring now to FIG. 8, in an alternate embodiment, system 800 includesa dehumidifying chamber 801, a drying chamber 803, and a sterilizationchamber 807. System 800 may additionally include hoses or connectors802. Drying chamber 803 may include one or more perforated dryingscreens or perforated shelves 805 for drying material to be dried 804.Sterilization chamber 807 may include an ultraviolet lamp 808 that emitsultraviolet radiation 809, which is used to sterilize the air. System800 may additionally include a fan 811 having blades 811 a and filter812. Once again, arrows indicate air flow.

As provided above, a system or apparatus for removing moisture frombiological material includes: a source of low-humidity air or dry inertgas, a first enclosure containing material to be dried (e.g., dryingchamber), a second enclosure containing means for sterilizing matter(e.g., sterilization chamber), means for moving the air through thefirst enclosure so as to evaporatively dry the material to be dried, andmeans for moving the air from the first enclosure through the secondenclosure so as to sterilize the air prior to release.

Benefits of the system for drying the biomass include:

-   -   No exposure to elevated temperatures, preserving stability of        heat-sensitive chemicals of interest;    -   Rapid drying due to thin-layer distribution of the biomass,        exposure to dehumidified air or dry gas, and reliance on        continuous air flow; rapid drying can also serve to stop cells'        metabolic activities, potentially preserving TAG stores.    -   Reduced energy expenditure by avoiding either heating or active        cooling of materials; and    -   Active control of the exhaust gas to prevent the spread of        undesirable organisms.

Because extracted liquids may contain residual nutrients, as well asmicrobial cells that escaped harvest, this fluid may be recycled. Forexample, in one embodiment, the recycled fluid constitutes a portion ofthe starting broth (e.g., liquid medium) of the next production cycle.Because the fluid may also contain metabolites released by thereproducing and digesting microbes, and high metabolite concentrationmay inhibit the succeeding production cycle, in one embodiment, therecycled fluid is treated to neutralize the metabolites. The recycledfluid may also, in some instances, be sterilized.

Following collection, the cellular biomass is exposed to a celldisruptor, e.g., means for extracting the lipid material from within thecells. In some embodiments, the cell disruptor frees lipids from microbecells using, for example, heat, cold, ultrasound or chemical disruption(lysis) of the cells. In one embodiment, chemical lysis includesutilizing a polar/non-polar solvent mix, such as a chloroform-methanolsolution to lyse the cells and their internal structures. Withoutwishing to be bound by any particular theory, it is believed that themethanol disrupts the cell, and the chloroform extracts the lipids.Other chemical solvents, including but not limited to methylene chlorideand chloroform-methanol, as well as hexane-ethanol and others, may alsobe used in chemical lysis and lipid extraction.

Once the lipids have been released from the intracellular structure,they are separated from the cellular debris. In some embodiments, amechanical lipid separator is used. For example, a doctor-blade to guidea floating lipid-rich mass from the top of the mixture, a sump to drawheavier components from the bottom of the lipid separator, or other portmeans depending on the properties of the lipids may be used.Furthermore, in some embodiments, a chemical solvation process may beutilized to provide a higher level of purity of TAG. For example, usinglight alkane solvents like hexane or heptanes yields a purer TAG thanmechanical means because phospholipids and proteins are typicallyinsoluble or slightly soluble in alkanes. Consequently, the resultingTAG may be low in contamination by phosphorus and metals, which isdesirable in some fuels.

After extraction of TAG, TAG is converted into hydrocarbons that maythen be fractionated to form constituents of gasoline, diesel or jetfuel. Such conversion process is known to those skilled in the art. TAGcan also be converted into alkyl esters such as methyl or ethyl to formbiodiesel, via transesterification, in conversion process known to thoseskilled in the art.

Referring now to FIG. 9, a flow chart of a microbial biomass orintermediary product collection process 900 in accordance with anembodiment of the invention is shown. The microbial collection process900 includes a receiving stage 910 for receiving the depleted fluid 540with suspended microbes containing TAG from the inoculation andfermentation process 500 and uses one or more separation technique asdescribed herein to harvest or collect 920 microbial biomass orintermediary product 930. In some embodiments, mechanical means such asone or more of filtration, sieving, screening, centrifugation orprecipitation, is used to separate the microbial biomass 930 from thedepleted liquid 925. Accordingly, in a preferred embodiment, it isadvantageous to use P. menonorum because its filamentatious structureenables the use of simple, inexpensive mechanical separators. Incontrast, non-aggregating unicellular microbes would require moreexpensive means, such as centrifuges.

In some embodiments, the depleted liquid 925 is recycled as part of thewater 134 added to the feedstock in the pretreatment stage 100 ofFIG. 1. The depleted liquid 925 may require buffering, not shown, tomitigate the otherwise inhibitory effect of metabolites secreted by themicrobes in the metabolization stage 530 of FIG. 5.

The microbial biomass or intermediary product 930 consists of wetmicrobial biomass. Accordingly, a drying step 940 may optionally beperformed, to speed the extraction process. The drying step 940 mayutilize heating in an oven, heating and evacuation in a vacuum oven,lyophilization, with or without use of a cryogenic liquid, or otherdesiccation means. The result of this step 940 is a dry cellular biomassor intermediary product 950.

Either the wet biomass 930 or the dry biomass 950 is then subjected to acell disruption step 960 that breaks up the cell structures to renderthe TAG accessible to chemical solvents. The cell disruption step 960may utilize methods including one or more of mechanical, thermal, orchemical methods. For example, mechanical disruption methods may includeone or more of ultrasonic, cutting, pressing, rolling or abrading means.Thermal methods may use heated air or microwave energy, among othermeans. Chemical means use one of several chemical agents, including butnot limited to chloroform, chloroform and methanol, or methylenechloride. The output of the cell disruption step 960 is a biomass withliberated TAG 970. Disrupting chemicals used in this step 960 may becaptured, recovered and reused in a closed-cycle system. The microbialcollection process 900 also includes a TAG extraction or initialpurification step 980. In some embodiments, TAG extraction is performedvia chemical solvation, using solvents including short-chain alkanessuch as hexane and heptanes. Solvation is followed by decantation,repeated as needed to achieve the required yield of TAG. The output ofthe TAG extraction step 980 is extracted and purified TAG 984, alongwith cellular debris 988. Solvents used in this step 980 may becaptured, recovered and reused in a closed-cycle system.

As stated above, the dry biomass contains the TAG within the microbialcells. The next step simultaneously disrupts the cell and extracts theTAG. In one embodiment, it relies on a mixture of solvents:

-   -   a. a polar organic solvent (such as methanol, ethanol,        isopropanol, or the like) to disrupt the cell structures and        also extract the lipids, and    -   b. a non-polar organic solvent (such as hexane, chloroform,        methylene chloride, or the like) to extract the lipids        efficiently.

In one embodiment, the solvent comprises a mixture of 10% methanol and90% chloroform, by volume. In another embodiment, the solvent comprisesa mixture of 10% ethanol and 90% hexane. Generally, the amount ofmethanol used can vary between 0% and 30% and the amount of ethanol usedcan vary between 0% and 30%. The nonpolar organic solvent completes thebalance (e.g, so that the solvent mixture adds to 100%). The percentagesneed not be precise.

If the dry biomass is dense and jerky-like, it may be pre-soaked in thesolvent mixture for several hours prior to the next step. If it isporous and fluffy, pre-soaking is not needed.

Cell disruption and TAG extraction proceeds by percolating hot solventmixtures repeatedly through an amount of thy biomass. In the laboratory,this can be accomplished by a Soxhlet apparatus. At an industrial scale,the Soxhlet apparatus may be replaced by a system that is more robustand more energy-efficient at large scale. The underlying chemicalprinciple remains the same: repeated exposure of the dry biomass to thehot pure solvents until nearly all the cells are disrupted and nearlyall the liquids including neutral lipid TAG has escaped the biomass andgone into solution. In the Soxhlet apparatus, heat is applied to areservoir of solvent, causing it to boil. The vapor rises until itcondenses in a condenser cooled just below the boiling point. Thecondensate drips into a vessel containing the dry biomass (e.g., insidea filter). The hot, pure (and hence chemically more active) solventlevel rises to submerge the biomass. In the Soxhlet process, the solventis not only hot, but pure, because it is recondensed from vapor.Consequently, as more material is dissolved and extracted from thebiomass, the material is collected in the reservoir and the solvent isboiled off, making the solvent pure when it again comes into contactwith the biomass.

A siphon at the top of the vessel completely drains the vessel back intothe solvent reservoir every time the liquid in the vessel reaches thetop of the bend in the siphon. The biomass, constrained inside thefilter, cannot flow with the draining fluid, so that the reservoir onlycollects fluid. This process can take several tens of minutes. Duringthis time, the solvent mixture is both breaking down the cell structuresand dissolving the TAG (and other intracellular molecules). When thevessel empties into the solvent reservoir, it now carries the solutewith it. The cycle ofevaporation—condensation—filling—dissolving—siphoning may be repeateduntil no further significant quantity of TAG is extracted from thebiomass.

In some embodiments, the reservoir contains lipids (e.g., TAG),other-biomolecules soluble in the polar solvent, and the solvent itself.An evaporation and distillation stage evaporates the solvent out of themixture and condenses it, recapturing the solvent for reuse. What nowremains in the reservoir is called crude TAG, since it may containimpurities.

A refining step includes treating the crude TAG in a solvent made ofshort-chain hydrocarbons such as heptane or mixtures of heptane withhexane or petroleum ether. One embodiment uses a 1:1 mixture of heptaneand low-boiling-point petroleum ether (with boiling point between 40° C.and 60° C.). In other embodiments, the ratio of the solvents in themixture can vary from about 1:100, about 1:10, or about 1:5 to about5:1, about 10:1, or about 100:1.

This organic mixture is then decanted off or centrifuged from theinsoluble residues and the organic layer can be washed with brine (NaClin water) or other alkali salt solution. TAG remains dissolved in thissolvent, while phospholipids, methanol, proteins and other impuritiessoluble in water are washed out. While not wishing to be bound by anyparticular theory, it is generally important to remove phospholipids, asmany transport fuel specifications require low levels of phosphorus inthe fuel.

In one embodiment, the brine wash is followed by a deionized water wash,which removes the NaCl. Following the water wash, the material may bedried using anhydrous sodium sulfate (Na₂SO₄) to remove residual water.Other analogous materials can also be used for the drying step. Thesodium sulfate is thereafter removed by filtration (e.g., using filterpaper in a sintered-glass filter funnel). The filtered liquid containingthe extracted TAG is heated to evaporate the petroleum solvents (e.g.,heptane and petroleum ether), which may be re-condensed for reuse. Thisprocess produces the purified TAG as an oily residue.

In one example embodiment, approximately 2 kg of wet biomass yields ≦1kg of dry biomass. As an example, approximately 6 L of nonpolar-polarsolvent mixture is used in the Soxhlet to extract the TAG. Theapproximately 6.1 L of TAG-solvent mixture is evaporated to yieldapproximately ≦0.1 L of TAG. Approximately 100 mL of heptane-petroleumether solvent and similar quantities of brine and deionized water, andapproximately 50 gm of sodium sulfate, anhydrous, are used to purify theTAG, yielding ≦0.1 L of final product. As can easily be appreciated, theabove numbers do not represent the best possible TAG yield, but aremerely examples of amounts that can be expected in a moderate-scalelaboratory implementation of the process.

In accordance with some embodiments, a method of producing TAG includesa number of steps, each of which are shown in FIG. 10A. In oneembodiment, a TAG extraction and purification process 1000 includes:starting with wet biomass in step 1005, optionally drying the biomass(not shown), optionally pre-soaking the dry biomass in a mixture ofnonpolar and polar organic solvents in step 1010, disrupting the cellstructures and extracting the TAG in step 1015 (e.g., via a Soxhlet-likeprocess using a mixture of alcoholic and non-polar organic solvents),evaporating the solvent mixture from the collected liquid to producecrude TAG in step 1020, mixing the crude TAG with a light-hydrocarbonsolvent in step 1025; decanting the organic layer off from the insolubleresidue in step 1030, washing the resulting mixture (organic layer) withbrine or other alkali salt solution and removing the water-solubleimpurities in step 1035, washing the resulting liquid in water to removethe salt in step 1040, removing water from the resulting liquid usingsodium sulfate, anhydrous, or other suitable material in step 1045;filtering the resulting liquid (e.g., using filter paper and asintered-glass filter funnel) in step 1050; and evaporating thelight-hydrocarbon solvents from the resulting liquid in step 1055,resulting in purified TAG in step 1060.

Referring now to FIG. 10B, a method of producing TAG, in accordance withanother embodiment, is shown. With the appropriate choice of microbe andculture nutrients, the crude TAG produced by the extraction process(using, for example, the ethanol-hexane solvent mixture) includesapproximately 10% phospholipids, approximately 5% free fatty acids, andthe remainder mostly TAG, with some mono-acyl-glycerides anddi-acyl-glycerides. Purification of the crude TAG removes phospholipidsand free fatty acids, to the extent specified by downstream processesthat upgrade TAG into fuel.

Similar to the method shown in FIG. 10A, TAG extraction and purificationprocess 1400 includes: starting with wet biomass in step 1405,optionally drying the biomass (not shown), optionally pre-soaking thedry biomass in a mixture of nonpolar and polar organic solvents in step1410, disrupting the cell structures and extracting the TAG in step 1415(e.g., via a Soxhlet-like process using a mixture of alcoholic andnon-polar organic solvents), evaporating the solvent mixture from thecollected liquid to produce crude TAG in step 1420.

Crude TAG is combined with a chosen acid in step 1425. Typically,phosphoric acid of 85% concentration is used (a concentration that isconveniently available from suppliers), and this material is added in avolume ratio of approximately 1:100, acid to crude TAG. The ratio mayvary by a factor of five in either direction. A citric acid solution ofapproximately 10% can be used as an alternative to phosphoric acid. Thephospholipids and acid form a salt that separates from the TAG in step1430. After mixing the acid and crude TAG, the material is centrifugedin step 1435 to enable removal of the bulk of the phospholipid salts.

Centrifugation is followed by a wash of the TAG with pure water in step1440, further separating impurities that are removed by anothercentrifugation. This water wash and centrifugation step may be repeated,if needed, as shown in step 1445.

The TAG at this point (step 1450) is believed to contain up toapproximately 5% or more free fatty acids. Some downstream processes canmake use of these acids and convert them to fuel, while others cannot.If the downstream process can use the free fatty acids, then the TAG isdeemed sufficiently purified and is delivered for conversion to fuel instep 1455. If not, the following additional purification steps areperformed.

A titration is performed on an aliquot of the TAG to quantify its freefatty acid concentration in step 1465. Using a procedure well known inthe art, the appropriate amount of sodium hydroxide is calculated, andthat amount is added to the TAG in step 1460. The sodium hydroxidecombines with the free fatty acids, yielding soap (the process is calledsaponification) in step 1465. The soap is removed via a centrifugationstep, as shown in step 1470.

Centrifugation is followed by a wash of the TAG with pure water in step1475, further separating impurities that are removed by anothercentrifugation in step 1480. This water wash and centrifugation step maybe repeated, if needed. The end result of this process is purified TAGdelivered for downstream conversion to fuel (shown in step 1485).

Referring now to FIG. 11, a biomass drying process 1100 is shown. Dryingprocess 1100 includes optional paths for no drying 1110 (resulting inuntreated wet biomass 1115), for mechanical removal of gross water only1120 (resulting in partially dried biomass 1125), for mechanical waterremoval followed by more thorough drying 1130 (resulting in dry biomass1135), and for thorough drying not preceded by mechanical water removal1140 (resulting in dry biomass 1135). Three means for thorough dryingare shown: lyophilizing 1150, using a vacuum oven 1170, and other dryingmeans 1160 (e.g., drying apparatus shown in FIGS. 7 and 8).

In some embodiments, maintaining the purity of the solvent, as is doneby the evaporation-recondensation cycle in the Soxhlet apparatus, isnot, in fact, necessary to obtain efficient extraction of the lipids. Asis easily appreciated, in the Soxhlet, the biomass remains stationaryinside a confining filter (thimble), and the solvent acts by diffusiveprocesses within the biomass. In such a diffusion-dominated extraction,it is important to keep the chemical kinetics favorable to continueddissolution of the lipids in the solvent. If the solvent were allowed toaccumulate dissolved lipids, the chemical kinetics of dissolution wouldbecome less favorable due to partition coefficients of the lipids in thesolvents, leading to limited efficiency of lipid extraction.

In an alternative embodiment, the biomass is not confined to bestationary inside a filter (e.g., such as in the Soxhlet apparatus), butis freely suspended, as a slurry, in the solvent. An agitator such as,for example, a rotary stirring mechanism moves the biomass in a solventbath. In some embodiments, the solvent is heated to a temperature rangetypically between about 50 and 100° C., or in general from about halfthe boiling point of the solvent mixture up to just below the boilingpoint of the solvent mixture.

Physical agitation ensures that the solvent reaches all portions of thebiomass. Generally, agitation makes the chemical kinetics of dissolutionmore favorable, so that extraction effectiveness may be achieved that isequivalent to the Soxhlet process. In effect, physical agitationreplaces the evaporation-condensation cycle; in both cases, the solventis heated to increase the kinetics of the dissolution reaction. Whilenot wishing to be bound by any particular theory, it is believed that byavoiding the two phase transition cycle required in the Soxhlet process,the heating-physical agitation method saves a very significant amount ofenergy and cost. For example, evaporation and condensation is neededonly at the final separation stage, and then only needs to be performedonce instead of many times over. While not wishing to be bound by anyparticular theory, it is also believed that the physical apparatusrequired for this physical agitation-based process is much lessexpensive than that required for the Soxhlet process, thus saving asignificant amount of capital expenditure.

Following the dissolution process, the lipid-laden solvent is separatedfrom the biomass via physical filtration. An evaporation stageevaporates the solvent; thus, the solvent is recaptured in its purestate to process the next batch of biomass and leaves the crude lipidextract to be processed further.

In some embodiments, the cellular debris 988 is sent to a gasifier andconsumed to produce on-site electricity and/or process heat. Thecellular debris 988 may also be used as part of the carbon andnon-carbon nutrients in the metabolization stage 530 of FIG. 5.Alternatively, the cellular debris 988 may be collected, processed andsold as other products, such as livestock feed.

As is easily appreciated, TAG produced in accordance with embodiments ofthe present invention may be used as a liquid fuel suitable fortransportation uses. In some embodiments, the fuel product includessaturated non-aromatic hydrocarbon molecules (e.g., alkanes and branchedalkanes) with molecular weights in a predetermined range (e.g., asrequired by vehicle engines).

Table 1 shows exemplary TAG produced by P. menonorum.

TABLE 1 Fatty acid profile of the TAG produced by P. menonorum FattyAcid Carbon Number of Abundance Common Name Chain Length Double Bonds(wt %) Myristic Acid 14 0 0.31 Pentadecylic Acid 15 0 0.21 Palmitic acid16 0 20.89 Margaric Acid 17 0 0.21 Stearic Acid 18 0 7.07 Oleic Acid 181 20.52 Linoleic acid 18 2 45.31 Linolenic Acid 18 3 3.50 Arachic Acid20 0 0.27 Behenic Acid 22 0 0.59 Lignoceric Acid 24 0 1.12

As shown in Table 1, the main components of this particular TAG productinclude linoleic acid, oleic acid, stearic acid and palmitic acid. Thecarbon chain length distribution in Table 1 indicates that any liquidtransportation fuel can be refined from the product, with reasonableefficiency.

As is easily appreciated, the product composition may be adjusted, byvarying process conditions, to partially offset feedstock variations andto meet application specifications. Depending on product specifications,in some embodiments, the liquid fuel product may contain a proportion ofsaturated aromatic carbon compounds. For example, jet fuelspecifications call for aromatic components comprising between 8% and25%, by weight, of the total fuel composition.

Filtration/Extraction Apparatus

In accordance with the above, in certain embodiments described herein,the filtration/drying chamber, cell disruption apparatus and TAGextraction apparatus described hereinabove comprise a cellulosedegrading fungus. In a preferred embodiment, the fungus comprises acellulose degrading fungus of the genus Penicillium. For example, thefiltration/drying apparatus may be a vessel as shown in FIGS. 7A, 7B and8. However, it will be readily appreciated that any suitablefiltration/drying system suitable for the extraction of moisture from awet mass of a cellulose degrading fungus can be used.

Similarly, the cell disruption and TAG extraction apparatus may be anapparatus as described hereinabove and in the process diagramed in FIG.10. However, it will be readily appreciated that any suitable celldisruption system or TAG extraction suitable for the disruption of acellulose degrading fungus or the extraction of TAG from a cellulosedegrading fungus can be used.

As used herein, the terms “filtration apparatus”, “drying chamber”,“filter”, “drying vessel”, “drying apparatus” and the like refer to anapparatus or chamber in which a mycelial mat of a cellulose degradingfungus such as a fungus of the genus Penicillium can be dispersed on afiltration surface such as a filter, screen, sheet and the like. Afiltration apparatus, such as any of those described herein and in theFigures, are useful for the extraction of moisture from a mass ofcellulose degrading fungi such as the fungus described herein asPenicillium menonorum. For example, in a filtration apparatus, any oneof a number of variables such as temperature, airflow, and the like maybe monitored and/or controlled to achieve a desired level of extractionof moisture. In a preferred embodiment, the mycelial mat is dried untilit reaches a constant weight. In other embodiments, the mycelial mat cancomprises less than 50% moisture, less than 45% moisture, less than 40%moisture, less than 35% moisture, less than 30% moisture, less than 25%moisture, less than 20% moisture, less than 15% moisture, less than 10%moisture, less than 5% moisture, less than 4% moisture, less than 3%moisture, less than 2% moisture and less than 1% moisture. It will bereadily appreciated that any suitable drying apparatus, including ascreen or sheet, can be utilized.

As used herein, the term “cell disruption apparatus” and the like refersto an apparatus in which the cellular structure of a cellulose degradingfungus such as a fungus of the genus Penicillium can be disrupted toresult in the liberation of cellular components such as TAG. A suitableapparatus may utilize methods including one or more of mechanical,thermal, or chemical methods. For example, mechanical disruption methodsmay include one or more of ultrasonic, cutting, pressing, rolling orabrading means. Thermal methods may use heated air or microwave energy,or for example, cryogenic means such as freezing in liquid nitrogen,among other means. Chemical means use one of several chemical agents,including but not limited to hexane and methanol, chloroform, chloroformand methanol, or methylene chloride. Accordingly, a cell disruptionapparatus, such as any of those described herein and in the Figures, isuseful for the liberation of TAG from a mass of cellulose degradingfungi such as the fungus described herein as Penicillium menonorum.

As used herein, the terms “extraction apparatus”, “TAG extractionapparatus” and the like refer to an apparatus in which the lipids,including TAG, from a cellulose degrading fungus, such as a fungus ofthe genus Penicillium, can go into solution. A suitable apparatus mayutilize the solvents and conditions described hereinabove. However, itwill be appreciated that any apparatus used to solubilize liberated TAGis useful as a TAG extraction apparatus. Accordingly, a TAG extractionapparatus, such as any of those described hereinabove, is useful for theextraction of TAG from a mass of cellulose degrading fungi such as thefungus described herein as Penicillium menonorum.

Accordingly, one embodiment described herein is a filtration apparatuscomprising a filter housing having a filtration surface disposedtherein, and a mycelial mat of a cellulose degrading fungus of the genusPenicillium dispersed on the filtration surface. In a preferredembodiment, the filtration apparatus comprises Penicillium menonorum.Preferably, the filtration apparatus comprises a fungus of the genusPenicillium, the species being the same as NRRL deposit Accession No:50410. In some aspects, the mycelial mat comprises a moisture content ofless than about 85% w/w. In other aspects, the mycelial mat comprises amoisture content of less than about 50% w/w. In other aspects, themycelial mat comprises a moisture content of less than about 25% w/w. Instill other aspects, the mycelial mat comprises a moisture content ofless than about 15% w/w. In yet other aspects, the mycelial matcomprises a moisture content of less than about 5% w/w.

Another embodiment described herein includes an extraction apparatuscomprising a vessel comprising a solvent and a fungus, wherein thesolvent is in contact with the fungus. In a preferred embodiment, theextraction apparatus comprises hyphal filaments of a cellulose degradingfungus of the genus Penicillium dispersed in an aprotic solvent. Typicalsolvents can include a polar organic solvent (such as methanol, ethanol,isopropanol, or the like) and a non-polar organic solvent (such ashexane, chloroform, methylene chloride, or the like). It will beappreciated that any suitable solvent or mixture of solvents that allowsfor extraction of TAG from disrupted hyphal filaments and other cells ofthe fungus can be used. In a preferred embodiment, the extractionapparatus comprises Penicillium menonorum. Preferably, the apparatuscomprises a fungus of the genus Penicillium, the species being the sameas NRRL deposit Accession No: 50410.

Another embodiment presented herein is an extraction apparatuscomprising a vessel comprising a solvent and a fungus, wherein thesolvent is in contact with the fungus. In a preferred embodiment, theextraction apparatus comprises hyphal filaments of a cellulose degradingfungus of the genus Penicillium dispersed in a solvent comprising lessthan 80% water. In some embodiments, the solvent comprises at least onealiphatic solvent. In some embodiments, the solvent comprises at leastone polar solvent. In certain embodiments, the polar solvent is aprotic.In some embodiments, the solvent comprises a non-polar organic solvent(such as hexane, chloroform, methylene chloride, or the like) and apolar organic solvent (such as methanol, ethanol, isopropanol, or thelike). In one preferred embodiment, the solvent comprises hexane andethanol. In a preferred embodiment, the alcohol-based solvent is at aconcentration of about 0%, 1%, 2%, 3%, 4%, 5%, 6,%, 7%, 8%, 9%, 10%,15%, 20%, 25% or about 30%, by volume. In a preferred embodiment, theethanol is at a concentration of about 10-20%. In a more preferredembodiment, the ethanol is at a concentration of about 10%. In anotherembodiment, the solvent comprises a mixture of methanol and chloroform.In a preferred embodiment, the apparatus comprises Penicilliummenonorum. Preferably, the apparatus comprises a fungus of the genusPenicillium, the species being the same as NRRL deposit Accession No:50410.

In a preferred embodiment, the fungus in the filtration apparatus orextraction apparatus described above can have a 5.8 S ribosomal RNA genesequence with sequence identity with the nucleic acid of SEQ ID NO: 1.In a preferred embodiment, the fungus can have an ITS1 sequence havingsequence identity with the nucleic acid of SEQ ID NO: 2. In a preferredembodiment, the fungus can have an ITS2 sequence having sequenceidentity with the nucleic acid of SEQ ID NO: 3. In a preferredembodiment, the fungus can have a 28 S rRNA gene sequence havingsequence identity with the nucleic acid of SEQ ID NO: 4. Morepreferably, the sequence identity with SEQ ID NOs: 1, 2, 3 and/or 4 isat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%nucleotide sequence identity with the nucleic acid of SEQ ID NO: 1, 2, 3and/or 4.

Extracting Aromatic Compounds

As stated above, extracting product from a digester is different,depending on whether the product is TAG from cellulose breakdown oraromatic hydrocarbons from lignin breakdown. The digester that receivesthe solid, lignin-rich portion of pretreated feedstock includes water,nutrients and an appropriate inoculum added to break the lignin downinto a variety of aromatic compounds. At the end of the fermentation ordigestion cycle, the solid mass is a combination of microbes andundigested solid feedstock, which can sometimes be waste.

The aromatic compounds are included as part of the liquid and gas phaseof the digester output (rather than being stored intracellularly as inTAG production). This is because the microbes break lignin down notprimarily to digest it for nutrient value, but to gain access toproteins inside the lignin structures. Thus, the microbes do not absorband metabolize the lignin breakdown products.

In some embodiments, the solid portion of the digester contents cansometimes be waste that can be disposed of or gasified to produceelectricity and process heat. Standard chemical separation andpurification processes may be implemented to capture the aromatics fromthe liquid and gas-phase outputs of the fermentation.

After extraction of the aromatic compounds, the aromatics may then befractionated by molecular weight. The fractionated aromatics may then beblended with alkanes to form constituents of gasoline, diesel or jetfuel. Such blending process is known to those skilled in the art.

Referring now to FIG. 12, a flow chart of a separation process 1200 inaccordance with an embodiment of the invention is shown. The separationprocess 1200 includes a receiving stage 1210 for receiving the mixture640 containing depleted solids, microbes, and gas and liquid containingthe desired aromatic compounds yielded by the metabolization step 630 ofFIG. 6.

The separation process 1200 subjects the mixture 640 to a mechanicalsolids separation step 1220. This separation step 1220 uses one or moreof standard mechanical means such as screening, sieving, centrifugationor filtration to achieve the separation. The separated depleted solidsand microbes 1225 can be sent to a gasifier and consumed to produceon-site electricity and/or process heat. Alternatively, the depletedsolids may be collected, processed and sold as other products, such aslivestock feed. In yet another embodiment, the separated depleted solidsand microbes 1225 may be further processed, such as via lipid extraction1250.

The separation step 1220 also outputs liquid and gas 1230 containing thetarget aromatic compounds. A chemical separation step 1240, usingstandard chemical processes known in the art, separates aromaticcompounds from the others and fractionates them by molecular weight,yielding the aromatic compounds of interest 1244. The byproduct of thischemical separation step 1240 is the waste gas and liquid 1248, whichmay contain microbial cell bodies. In some embodiments, this wasteliquid 1248 is recycled to form part of the input water mixture 134 ofthe feedstock pretreatment stage 130 of FIG. 1.

The production of TAG and aromatic compounds may be associated with orimplemented by a cellulose processing plant and/or a bio-refineryproducing transportation fuel. The association may be integral,parallel, or separate.

In some embodiments, a cellulose processing plant receives agriculturalwaste (or other cellulosic material including animal manure), convertsit into TAGs by microbial action, and then extracts intermediates fromTAGs that may be converted to fuel. In contrast, a bio-refinerytypically receives TAG and aromatic compounds, processes them and blendsthem into transportation fuels.

In one embodiment, the production of TAG and aromatic compounds isimplemented by a cellulose processing plant in parallel with abio-refinery. In such an embodiment, glycerol produced by thebio-refinery is used to generate further lipids, and then either convertthe lipids into fuel or pass the lipids to the bio-refinery plant whichconverts the lipids to fuel.

In another embodiment, the production of TAG and aromatic compounds isimplemented by a cellulose processing plant integrated with abio-refinery. In such an embodiment, the cellulose processing system isutilized to produce glycerol. For example, the same vessel may containboth the cellulose digestion mixture and the glycerol consumptionmixture intermingled. The microbes for cellulose digestion and glycerolconsumption may be intermingled if they are compatible. It will beappreciated that the same microbe may perform both cellulose digestionand glycerol consumption simultaneously. Similarly, a single combinedlipid product may be recovered from both processes.

In another embodiment, the production of TAG and aromatic compounds isimplemented by a cellulose processing plant separate from abio-refinery. In such an embodiment, the glycerol processing is separatefrom the cellulose processing. In one example, the glycerol feed may bereduced all the way to the fuel product. Alternatively, the glycerolfeed may provide lipids as an intermediate product, with fuel productionbeing completed at the separate bio-refinery or chemical refinery. Insome embodiments, alkanes are extracted from TAGs and recycled in theglycerol processor to generate further fuel. This process may berepeated in cyclical fashion until the feed material is exhausted.

From the above description, a method in accordance with embodiments ofthe present invention, include a series of steps. These steps includeone or more of the following:

-   -   (1) Receiving and pretreating cellulosic feedstock;    -   (2) Optionally, adding glycerol obtained as a co-product of TAG        transesterification;    -   (3) Separating the pretreated feedstock into liquid and solid        phases;    -   (4) Inoculating the liquid phase with microbes that are capable        of converting the carbon into lipids, then allowing the microbes        to do so;    -   (5) Harvesting the resulting microbial biomass from the liquid;    -   (6) Extracting the lipids for subsequent conversion into fuels.    -   (7) Mixing the solid-phase pretreated feedstock with water and        nutrients, then inoculating it with microbes capable of        attacking the lignin and converting it into aromatic compounds;    -   (8) Separating the resulting aromatics from the liquid and gas        phases of the digester output;    -   (9) Recycling the remaining solid-phase matter as a co-product        or as feed for gasification and conversion to heat and        electricity or as added carbon feedstock for subsequent        microbial processing;    -   (10) Recycling the liquid-phase matter as broth for the next        batch of feedstock and fermentation.

Additionally, in some embodiments, the pretreatment process 100, asshown in FIG. 1, leaves considerable cellulose and hemicellulose in thesolid phase or portion 148. In such embodiments, the solid-phasefeedstock 148 is inoculated with a consortium of microbes that includesspecies to digest the cellulose and hemicellulose and produceintracellular TAG as well as species to break down the lignin andsecrete aromatic molecules in step 620. Following the metabolizationstep 630, the aromatic compound separation 1220 proceeds as indicated inFIG. 12, but the solid phase extract 1225 is no longer mere waste orrecycling material, but is subjected to the TAG extraction process 980of FIG. 9.

In some embodiments, the liquid-solid separation step 140 at the end ofthe feedstock pretreatment process 100 of FIG. 1 is absent. In suchembodiments, the unseparated feedstock is inoculated with a consortiumof microbes capable of digesting both liquid and solid phases, thearomatic compounds are separated as shown in FIG. 12, and the TAG isextracted as shown in FIG. 9.

Turning now to FIG. 13, a system 1300 for producing TAG in accordancewith an embodiment is shown. System 1300 includes a processing plant orfacility 1310 in communication with a controller 1390. In oneembodiment, processing plant 1310 communicates with controller 1390 viaa network connection 1380. Network connection 1380 may be wireless orhard-wired. Network connection 1380 may also include the use of a webbrowser or other internet connectivity to allow observation and controlfrom a remote location.

In some embodiments, controller 1390 provides operating instructions forprocessing plant 1310's operating conditions. Controller 1390 mayreceive information from processing plant 1310 and utilize theinformation as feedback to adjust operating instructions to processingplant 1310. Parameters that can be actively controlled in this wayinclude, among others, temperature, pH, dissolved oxygen, and thecontrolled continuous feed or carbon and/or carbon nutrients.

In one embodiment, the operating conditions may be presented on amonitor or display 1395 and a user may interact with the operatingconditions via a user interface. The monitor 1395 may be in the form ofa cathode ray tube, a flat panel screen or any other display module. Theuser interface may include a keyboard, mouse, joystick, write pen orother device such as a microphone, video camera or other user inputdevice.

Processing facility 1310 includes sterilization process equipment orsterilizer 1320, solids extraction process equipment or solids extractor1330, bioreactor 1340 (e.g., fermentation process equipment orfermentor), microbial biomass extraction process equipment or microbialbiomass extractor 1350, cell disruption process equipment or celldisruptor 1360 and TAG extraction and purification process equipment orTAG extractor 1370. In one embodiment, cell disrupter 1360 is microbialbiomass process equipment. In some embodiments, controller 1390 is incommunication with fermentor 1340 and provides/controls the operatingconditions of fermentor 1340.

Sterilization process equipment 1320 and solids extraction processequipment 1330 together perform the cellulosic feedstock pretreatmentprocess 100 of FIG. 1. Fermentation process equipment 1340 performs theinoculation and fermentation process 500 of FIG. 5. Microbial biomassextraction process equipment 1350, cell disruption process equipment1360 and TAG extraction process equipment 1370 together perform themicrobial biomass collection process 900 of FIG. 9.

Those of skill will appreciate that the various illustrative logicalblocks, modules, and algorithm steps described in connection with theembodiments disclosed herein can often be implemented as electronichardware, computer software, or combinations of both. To clearlyillustrate this interchangeability of hardware and software, variousillustrative components, blocks, modules, and steps have been describedabove generally in terms of their functionality. Whether suchfunctionality is implemented as hardware or software depends upon thedesign constraints imposed on the overall system. Skilled persons canimplement the described functionality in varying ways for eachparticular application, but such implementation decisions should not beinterpreted as causing a departure from the scope of the invention. Inaddition, the grouping of functions within a module, block or step isfor ease of description. Specific functions or steps can be moved fromone module or block without departing from the invention.

The various illustrative logical blocks and modules described inconnection with the embodiments disclosed herein can be implemented orperformed with a general purpose processor, a digital signal processor(DSP), application specific integrated circuit (ASIC), a fieldprogrammable gate array (FPGA) or other programmable logic device,discrete gate or transistor logic, discrete hardware components, or anycombination thereof designed to perform the functions described herein.A general-purpose processor can be a microprocessor, but in thealternative, the processor can be any processor, controller,microcontroller, or state machine. A processor can also be implementedas a combination of computing devices, for example, a combination of aDSP and a microprocessor, a plurality of microprocessors, one or moremicroprocessors in conjunction with a DSP core, or any other suchconfiguration.

The steps of a method or algorithm described in connection with theembodiments disclosed herein can be embodied directly in hardware, in asoftware module executed by a processor, or in a combination of the two.A software module can reside in RAM memory, flash memory, ROM memory,EPROM memory, EEPROM memory, registers, hard disk, a removable disk, aCD-ROM, or any other form of storage medium. An exemplary storage mediumcan be coupled to the processor such that the processor can readinformation from, and write information to, the storage medium. In thealternative, the storage medium can be integral to the processor. Theprocessor and the storage medium can reside in an ASIC.

Facilities and Processes for Producing Biofuels and Co-Products

As described hereinabove, the cellulose degrading fungus of the genusPenicillium can be grown in a bioprocess reactor under conditions thatpermit the fungus to produce biofuel and one or more economicallyvaluable co-products. Some economically valuable products andco-products include, for example, lipids, aromatic compounds and otherbreakdown products, conditioned medium, residual solids derived fromdigested feedstock, metabolites, phospholipids, and spent fungal cells.Each of these co-products is discussed briefly below.

The main product of this process is lipids sequestered inside the fungalcells and extracted via the processes described hereinabove. The lipidsare primarily tri-acyl-glycerides (TAG), and their fatty acid componentshave a carbon chain length distribution well suited to subsequentprocessing into fuel (whether biodiesel as fatty acid methyl esters orpetroleum replacement fuels as hydrocarbons). The TAG forms anywherefrom 5% to more than 35-40% of the total dry weight of the fungal cells.

Metabolites with Pharmaceutical Properties

The liquid culture medium contains metabolites produced by the fungusduring its life cycle. These metabolites may include co-products whichare useful as pharmacologically active compounds such as antibiotics,antifungals, anti-cancer agents, anti-atherosclerotic agents, and thelike.

Accordingly, presented herein is a conditioned medium comprising asubstance that inhibits the growth of an organism, wherein the medium isconditioned by a cellulose degrading fungus. Also presented herein is avessel comprising an antimicrobial substance obtained from a cellulosedegrading fungus of the genus Penicillium. In preferred embodiments, themedium is conditioned by Penicillium menonorum. Preferably, the mediumis conditioned by a fungus of the genus Penicillium, the species beingthe same as NRRL deposit Accession No: 50410.

In one preferred embodiment, the medium is conditioned by a fungushaving a 5.8 S ribosomal RNA gene sequence with sequence identity withthe nucleic acid of SEQ ID NO: 1. In a preferred embodiment, the funguscan have an ITS1 sequence having sequence identity with the nucleic acidof SEQ ID NO: 2. In a preferred embodiment, the fungus can have an ITS2sequence having sequence identity with the nucleic acid of SEQ ID NO: 3.In a preferred embodiment, the fungus can have a 28 S rRNA gene sequencehaving sequence identity with the nucleic acid of SEQ ID NO: 4. Morepreferably, the sequence identity with SEQ ID NOs: 1, 2, 3 and/or 4 isat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%nucleotide sequence identity with the nucleic acid of SEQ ID NO: 1, 2, 3and/or 4.

As used herein, an inhibitory substance can be any pharmacologicallyactive compound that inhibits the growth of an organism. In someembodiments, the inhibitory substance inhibits the growth of eukaryoticorganisms. In some embodiments the inhibitory substance inhibits thegrowth of prokaryotic organisms. Thus, in some embodiments, theinhibitory substance is an antimicrobial substance. For example, theinhibitory substance can inhibit the growth of eubacteria,archeabacteria or both. The bacteria can be, for example gram positiveor gram negative bacteria. Where the inhibitory substance is anantibacterial agent, the substance can be, for example, a bacteriostaticagent and/or a bacteriocidal agent.

It will be appreciated that the inhibitory substances described hereinare useful, for example, for preventing contamination or infection witha microbe. It will also be appreciated that the inhibitory substancesdisclosed herein are useful in treating contaminated materials ororganisms infected with one or more microbes.

Embodiments of the present disclosure also include processes forproducing an inhibitory substance produced by a cellulose degradingfungus. In some embodiments, the process can comprise, for example: a)incubating a cellulose degrading fungus of the genus Penicillium in aliquid medium for a sufficient time for production of an inhibitorysubstance by the fungus; and b) separating the fungus from the liquidmedium, thereby producing a harvested fungus and a conditioned liquidmedium, wherein the conditioned liquid medium comprises the inhibitorysubstance produced by the fungus.

In some embodiments, the inhibitory substance can be furtherconcentrated, either before or after separating the fungus from theliquid medium. Methodologies for concentrating a substance in solutionare well-known in the art, and include such methodologies as dialysis,ultrafiltration, evaporation, distillation, liquid chromatography andthe like.

Extracting Co-Products from Conditioned Medium

In some embodiments, the process for producing an inhibitory substancecan further comprise: extracting the inhibitory substance from theconditioned medium. In certain embodiments, the conditioned medium hasbeen fully separated from the harvested fungus. In certain embodiments,not all of the fungus has been separated from the conditioned medium.Methods of extracting compounds from a liquid are well known in the art,and include, for example, liquid-liquid extraction. It will beappreciated that any suitable solvent or mixture of solvents that allowsfor extraction of an inhibitory substance from the conditioned mediumcan be used. In some embodiments, a solvent that is not miscible withthe conditioned medium is used. It will be appreciated that one of skillin the art will be able to select a solvent ranging from a non-polar tosolvents of increasing polarity.

In preferred embodiments, the extraction solvent can be one or moreorganic solvents. The term “organic solvent” as used herein refers toany carbon-based organic solvent. In certain embodiments, the organicsolvents range from simple alkanes to functionalized hydrocarbons. Forexample, the organic solvent can include, but is not limited to,propane, butane, pentane, hexanes, heptanes, other alkanes and allassociated isomers, esters, ethers, ketones, acetates, and combinationsand blends thereof in any suitable ratio. It will be appreciated thatthe organic solvent can be chosen so as to adjust the polarity of thesolvent in order to obtain more pure fractions. Thus, for example, sometypical solvents include, but are not limited to: hexane, methy t-butylether, methylene chloride, ethyl acetate, amyl acetate,isopropanol/methylene chloride mixture, ethyl acetate at acidic pH(e.g., pH 2.5), amyl acetate at acidic pH (e.g., pH 2.5) and mixturesthereof. In preferred embodiments, the organic solvent is liquid at theoperating pressures and temperatures of the extraction processespresented herein.

In certain embodiments, the inhibitory substance is extracted directlyfrom the conditioned medium or a concentrated form of the conditionedmedium. In other embodiments, the inhibitory substance is extracted fromthe conditioned medium after a series of extractions, each subsequentextraction performed on the conditioned medium. In other embodiments,the inhibitory substance is subjected to a series of extractions, eachsubsequent extraction performed on the extraction solvent of theprevious extraction.

In some embodiments, the extraction solvent is not suitable for otherdownstream applications including, for example, analysis of theinhibitory substance and/or treatment of an animal with the inhibitorysubstance. Thus, in certain embodiments, the extraction solvent can beevaporated or otherwise removed from the inhibitory substance. Theinhibitory substance can then be reconstituted in some other solventthat is suitable for the downstream application.

Accordingly, also presented herein is an extract of medium conditionedby a cellulose degrading fungus of the genus Penicillium, the extractcomprising one or more compounds having antimicrobial activity. Incertain embodiments, the extract comprises one or more hexane solublecompounds. In certain embodiments, the extract comprises one or moremethy t-butyl ether soluble compounds. In certain embodiments, theextract comprises one or more methylene chloride soluble compounds. Incertain embodiments, the extract comprises one or more ethyl acetatesoluble compounds. In certain embodiments, the extract comprises one ormore isopropanol:methylene chloride (20%:80%) soluble compounds. Incertain embodiments, the extract comprises one or more ethyl acetate (atpH 2.5) soluble compounds. Specific examples of extracts containing aninhibitory substance are provided in greater detail in Examples 9 and 10below.

In preferred embodiments, the extract is derived from medium conditionedby Penicillium menonorum. Preferably, the medium is conditioned by afungus of the genus Penicillium, the species being the same as NRRLdeposit Accession No: 50410. In one preferred embodiment, the medium isconditioned by a fungus having a 5.8 S ribosomal RNA gene sequence withsequence identity with the nucleic acid of SEQ ID NO: 1. In a preferredembodiment, the fungus can have an ITS1 sequence having sequenceidentity with the nucleic acid of SEQ ID NO: 2. In a preferredembodiment, the fungus can have an ITS2 sequence having sequenceidentity with the nucleic acid of SEQ ID NO: 3. In a preferredembodiment, the fungus can have a 28 S rRNA gene sequence havingsequence identity with the nucleic acid of SEQ ID NO: 4. Morepreferably, the sequence identity with SEQ ID NOs: 1, 2, 3 and/or 4 isat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%nucleotide sequence identity with the nucleic acid of SEQ ID NO: 1, 2, 3and/or 4.

Phopholipids and Other Co-Products

Phospholipids are often present in the TAG extract. As extracted fromthe cells, phospholipids are present as anywhere from ˜1% to ˜30% of thecrude extract. These are typically separated from the actual TAG in asubsequent purification process; when the phospholipids themselves arepurified, they form lecithin, a co-product.

The spent fungal cells, after the TAG has been extracted, containproteins that can form a co-product: a nutrient for land and aquaticspecies raised as food for humans, and/or as pets. The material can alsofind use as a human foodstuff or ingredient thereof.

Accordingly, facilities made up of a plurality of bioprocess reactorsare useful as a means to increase production of the biofuels and otherco-products. As such, described herein is a bioprocessing facility forproducing co-products, the facility comprising a plurality of bioprocessreactors. The plurality of bioprocess reactors can vary in size andcapacity in order to allow scale-up of a culture, for example. Thebioprocess reactors as described hereinabove can comprise a cellulosedegrading fungus of the genus Penicillium growing in a liquid medium,the liquid medium comprising a carbon source and a nitrogen source,wherein the ratio of carbon to nitrogen in the medium ranges from about1:1 to about 1000:1. In typical embodiments, the fungus comprisesPenicillium menonorum. In preferred embodiments, the fungus comprises aspecies being the same as NRRL deposit Accession No: 50410.

In accordance with the above, also described herein is a process formanufacturing a plurality of co-products, the process comprising: a)inoculating a bioprocess reactor with an inoculum of a cellulosedegrading fungus of the genus Penicillium; b) allowing a sufficient timefor production of TAG by said fungus, wherein said TAG comprises atleast 5% of the dry weight of the fungus; c) harvesting said fungus; d)extracting lipids from said fungus, thereby producing a lipidco-product. The process can further comprise e) drying said fungus,thereby producing a feed co-product. In some aspects, the TAG comprisesat least 35% of the dry weight of the fungus. In other aspects, thelipid co-product comprises a TAG co-product and a phospholipidco-product. In still other aspects, the phospholipid co-productcomprises lecithin. In yet other aspects, the feed co-product issuitable for animal and/or human consumption. In typical embodiments,the fungus comprises Penicillium menonorum. In preferred embodiments,the fungus comprises a species being the same as NRRL deposit AccessionNo: 50410.

In some embodiments, one or more co-products are harvested from thereaction vessel to separate the one or more co-products from the otherremaining components. Co-products which can be harvested include, forexample, lipids, aromatic compounds and other breakdown products,conditioned medium, residual solids derived from digested feedstock,metabolites, phospholipids, and fungal biomass. The one or moreco-products can be harvested separately, or they can be harvestedtogether. The one or more co-products can be harvested simultaneously,in sequence, or separately. After harvesting a component or co-product,the one or more co-products may need to be further extracted, asdescribed elsewhere herein.

In some embodiments, the bioprocess reactor comprises a carbon sourceselected from the group consisting of: a solid feedstock, a lysate in aliquid, a gaseous vapor, and a combination thereof. Thus, for example,in embodiments where lignin is present in solid feedstock, aromaticcompounds can be harvested from the reaction medium. In certainembodiments, the aromatic compounds are further extracted from themedium.

Furthermore, also described herein is a facility comprising: a vesselfor holding liquid medium conditioned by a cellulose degrading fungus ofthe genus Penicillium; an extraction apparatus comprising an extract oflipids derived from said fungus; and a drying apparatus comprising driedfungus. It will be appreciated that some metabolites produced by thecellulose degrading fungus are secreted by the organism into the liquidmedium. As such, the vessel can contain one or more metabolites that areuseful as co-products. The extraction apparatus can comprise extractionsolvent that contains lipids extracted from the cellulose degradingfungus. Typically, the extract comprises TAG. Additionally, the extractof lipids can comprise phospholipids, such as, for example,phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositoland the like. When such phospholipids themselves are purified, they canform lecithin. The drying apparatus can comprise dried biomass,comprising spent fungal cells after the TAG has been extracted. Thedried biomass typically contains proteins, carbohydrates, and fiber thatare suitable, for animal and/or human consumption. In typicalembodiments, the fungus comprises Penicillium menonorum. In preferredembodiments, the fungus comprises a species being the same as NRRLdeposit Accession No: 50410.

Improved Animal Feeds

Presented herein is the discovery that feed formulations incorporatingfungal biomass have superior qualities and that animals fed using suchformulations may have increased survival compared to existing feeds.Also presented herein is the discovery that feed formulationsincorporating fungal biomass have at least equivalent if not superiorqualities to standard feed formulations that do not incorporate fungalbiomass and that animals fed using such formulations have at leastequivalent if not increased survival compared to existing feeds. Alsodisclosed herein is a new animal meal formulation that also provides amarket for whey, which had been a waste product from a differentindustry: cheese manufacture.

Another aspect of this disclosure is the admixture of the microbialbiomass, following extraction of the lipids comprising fuel precursors,with whey. Whey liquids are a waste product resulting from themanufacture of cheese. The volume of this waste product is significant,as it constitutes approximately 85-90% of the milk volume, and retains55% of milk nutrients. Among the most abundant of these nutrients arelactose (3.8-5.0% w/v), soluble proteins (0.6-0.8% w/v), lipids, andmineral salts. The waste must be processed in sewage treatment plants,and many jurisdictions impose significant costs on the cheese producersin order to process the material. Accordingly, whey disposal representsa major expense in cheese production. The composition of whey variesfrom process to process, but represents approximately 3.8-5% w/w oflactose or lactate, and 0.4-0.8% w/w of proteins. By combining the wheywith the dried microbial animal meal, a new product is formed withbeneficial nutritive properties. This renders the whey, heretofore awaste creating disposal expense, into a co-product representing a sourceof revenue to the cheese producer.

Contemplated herein are compositions that include one or more specificgenera and species of microbes that not only convert a wide range ofcellulosic feedstocks into lipids effectively, but also yield desirableco-products. An example of the latter includes the spent microbialbiomass after the lipids have been extracted, which can be sold asprotein meal to feed livestock in agriculture, aquaculture or pets. Theprotein meal may also find use as a source of human nutrition. Primaryor secondary metabolites produced and/or secreted by the one or moremicrobes provide another class of co-products. In some embodiments, themetabolites may be primary metabolites.

As described hereinabove, the cellulose degrading fungus of the genusPenicillium can be grown in a bioprocess reactor under conditions thatpermit the fungus to produce biofuel and one or more economicallyvaluable co-products. Some economically valuable products andco-products include, for example, lipids, metabolites, phospholipids,and spent fungal cells. The discussion below focuses on the last-namedco-product.

The spent fungal cells, after the TAG has been extracted, containproteins that can form a co-product: a nutrient for land and aquaticspecies of animals raised as food for humans, and/or as pets. Thematerial can also find use as a human foodstuff or ingredient thereof.

Accordingly, facilities made up of a plurality of bioprocess reactorsare useful as a means to increase production of the biofuels and otherco-products. As such, described herein is a bioprocessing facility forproducing co-products, the facility comprising a plurality of bioprocessreactors. The plurality of bioprocess reactors can vary in size andcapacity in order to allow scale-up of a culture, for example. Thebioprocess reactors as described hereinabove can comprise a cellulosedegrading fungus of the genus Penicillium growing in a liquid medium,the liquid medium comprising a carbon source and a nitrogen source,wherein the ratio of carbon to nitrogen in the medium ranges from about1:1 to about 1000:1. In typical embodiments, the fungus comprisesPenicillium menonorum. In preferred embodiments, the fungus comprises aspecies being the same as NRRL deposit Accession No: 50410.

Animal Feed Compositions

Provided herein are animal feed compositions comprising a fungal biomassderived from a cellulose degrading fungus of the genus Penicillium. Theanimal feed compositions provided herein can comprise the fungal biomassin an amount ranging from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% to about 100% by weight. In some embodiments, the animalfeed can comprise the biomass in an amount ranging from 5% to 10% byweight. In typical embodiments, the fungus comprises Penicilliummenonorum, and related strains as described hereinabove and in theincorporated materials of Application No. 61/372,828, filed Aug. 11,2010, entitled, “Bioreactors Comprising Fungal Strains.” In preferredembodiments, the fungus comprises a species being the same as NRRLdeposit Accession No: 50410.

In certain embodiments provided herein, the animal feed has beensubstantially depleted of a lipid product. As used herein, the term“substantially depleted” refers to a significant depletion of the lipidproduct from the fungal biomass. In certain embodiments, substantialdepletion refers to depletion of more than 20%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95% or more than 99% of the lipid product. The lipidproduct can be, for example TAG. In some embodiments, the lipid productcan comprise TAG and a phospholipid co-product such as lecithin.

As described in Example 6 below, it has been surprisingly discoveredthat providing an animal feed comprising a fungal biomass derived from acellulose degrading fungus of the genus Penicillium may result inunusual and significant enhanced survival, indicating that such feedformulations can be in many ways superior to existing premium feeds.Accordingly, the animal feed formulations presented herein can beutilized in methodologies for improving the longevity of an animal. Insome embodiments, the animal feed provided herein performs at least aswell as, or superior to animal feed formulations currently known in theart for the purpose of improving animal growth and survival.

Additionally provided herein is the surprising finding that providing ananimal feed comprising a fungal biomass derived from a cellulosedegrading fungus of the genus Penicillium can result in a significantincrease in the resistance of an animal to an infection. Accordingly,methods for enhancing the resistance of an animal to infection areprovided. The infection can be, for example, a viral infection, abacterial, fungal or other microbial infection, or some other parasiticinfection. It will be appreciated that in embodiments where the animalfeed provided herein enhances the animal's immune system, the method cancomprise enhancing the animal's resistance to any infection that istargeted by the animal's immune system. In some embodiments, the animalfeed provided herein performs at least as well as, or superior to animalfeed formulations currently known in the art for the purpose ofimproving resistance to infection.

In embodiments provided herein, the animal feed comprising a fungalbiomass derived from a cellulose degrading fungus of the genusPenicillium is suitable for animal and/or human consumption. In someembodiments, the animal feed is suitable for consumption by a landanimal. Land animals are any animals that live predominantly on land andinclude, for example, cows, chickens, pigs, cats, dogs, humans, rodents,and the like. In some embodiments, the animal feed is suitable forconsumption by aquatic animals. Aquatic animals include, for example,fish, shrimp, prawns, aquatic mammals, and the like.

Animal Meal Including Whey

A major waste stream in cheese production is liquid whey. Municipalitiesoperating sewage treatment facilities often charge cheese producers afixed amount per gallon of whey poured into the sewage stream. Thus,handling waste whey becomes a significant expense in cheese production.The present invention discloses a means to treat the whey such that itcan become a revenue source rather than an expense. At the same time,the treatment removes the burden of treating whey in sewage treatmentfacilities.

Accordingly, presented herein is the discovery that whey can be mixedwith fungal cell residual biomass to form a protein meal slurry. Thissolves several problems. First, it combines the proteins and othernutrients of the fungal biomeal with those of the whey, adding to thenutritional value of the product. Second, it changes the consistency ofthe dry, granular fungal biomeal into a slurry that can be pumped intotanker trucks for transport, and thence into feeding stations fordistribution to farm animals. This, in turn, can reduce the cost ofdairy operations: the same tanker trucks that transport milk from thedairy farm to the cheese producer can now, instead of returning emptyfor the next load of milk, return carrying food for the dairy animals.Example 7, below, lists properties measured for mixtures of whey andfungal-cell biomeal.

In some embodiments, an admixture is provided comprising fungal biomassand whey. In some aspects, the fungal biomass comprises biomass derivedfrom a cellulose degrading fungus of the genus Penicillium. In someembodiments, the biomass and the whey form a slurry. In certain aspects,the biomass and whey can be in a dry biomass/whey ratio ranging fromabout 1:100 to about 100:1 (vol/vol). The ratio of dry biomass/whey canbe, for example, about 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30,1:20, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1,30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or about 100:1. In typicalembodiments, the biomass and whey are in a dry biomass/whey ratioranging from about 1:10 to about 10:1 (vol/vol). Thus, for example, inone embodiment of the invention, 0.17 liters of dry fungal-cell biomealare mixed with 0.5 liters of whey to produce a slurry capable of beingpumped with standard equipment into and out of tanker trucks such asmilk tankers. In other embodiments, between 1 liters of fungal-cellbiomeal can be combined with with between 4 and 5 liters of whey for thesame purpose.

In typical embodiments, the fungus comprises Penicillium menonorum, andrelated strains as described hereinabove. In preferred embodiments, thefungus comprises a species being the same as NRRL deposit Accession No:50410.

In certain embodiments provided herein, the animal feed has beensubstantially depleted of a lipid product. As used herein, the term“substantially depleted” refers to a significant depletion of the lipidproduct from the fungal biomass. In certain embodiments, substantialdepletion refers to depletion of more than 20%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95% or more than 99% of the lipid product. The lipidproduct can be, for example TAG. In some embodiments, the lipid productcan comprise TAG and a phospholipid co-product such as lecithin.

Accordingly, also provided herein are a methods of manufacture of ananimal feed comprising combining dried fungal biomass with whey, therebyforming an admixture. In one embodiment, the method comprises, prior tocombining: extracting a lipid product from fungal biomass; and dryingthe fungal biomass, thereby producing dried fungal biomass.

Also provided, is a method of feeding an animal comprising providing tothe animal an admixture comprising fungal biomass and whey. In someaspects, the method further comprises identifying an animal that wouldlikely benefit from consuming the admixture. For example, identifying ananimal that would likely have a superior rate of growth or enhancedimmune system function if fed the fungal biomass or admixture describedherein as compared to if fed another feed. In some aspects, the fungalbiomass comprises biomass derived from a cellulose degrading fungus ofthe genus Penicillium.

In certain aspects, the biomass/whey admixture is suitable for animaland/or human consumption. In embodiments provided herein, the animalfeed comprising a fungal biomass derived from a cellulose degradingfungus of the genus Penicillium is suitable for animal and/or humanconsumption. In some embodiments, the animal feed is suitable forconsumption by a land animal. Land animals are any animals that livepredominantly on land and include, for example, cows, chickens, pigs,cats, dogs, humans, rodents, and the like. In some embodiments, theanimal feed is suitable for consumption by aquatic animals. Aquaticanimals include, for example, fish, shrimp, prawns, aquatic mammals, andthe like.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the teachings herein.

Example 1 Identification and Sequencing

A region of the genome of strain MM-P1 was sequenced to allow comparisonto sequences in the National Center for Biotechnology Information (NCBI)nucleotide database. The region included the internal transcribed spacer1 (ITS1), the 5.8 S ribosomal RNA (5.8 S rRNA) gene, the internaltranscribed spacer 2 (ITS2), and part of the 28 S ribosomal RNA (28 SrRNA) gene. The identified sequence is set forth in FIG. 14.

A section of this sequence (773 nucleotides) was run through the NCBIBlastn program to identify related sequences in the NCBI nucleotidedatabase. The most similar sequence (98% identical) was that of the sameregion in Penicillium pimiteouiense. Because the MM-P1 sequence was notidentical to any other sequence in the database, it was considered topotentially be a new species.

Example 2 Comparison to Related Organisms

The ribosomal sequence of strain MM-P1 was compared to the correspondingribosomal sequences from other closely related fungi to determine thephylogeny of MM-P1. The nucleotide sequences corresponding to the ITS1,5.8 S rRNA, ITS2, and partial 28 S rRNA regions of these species andother Penicillium and Eupenicillium species were obtained from NCBI.Using the computer software, Geneious (version 5.0), the nucleotidesequences were assembled into a phylogenetic tree. FIG. 15 shows adendrogram of MM-P1 and its near neighbors, as determined by parsimonyanalysis. Numbers above the lines indicate bootstrap values for thosenodes; only bootstrap values over 70% are shown. The dendrogram setforth in FIG. 15 shows that MM-P1 was closely related to P.pimiteouiense and suggested some other close relatives to be used forcomparison purposes, including P. griseolum, P. striatisporum, P.vinaceum, Eupenicillium parvum, and E. rubidurum. The conclusion fromthese studies is that MM-P1 is most closely related to E. rubidurum, P.pimiteouiense, P. vinaceum, E. parvum, and P. striatisporum.

Example 3 Relationship of Toxigenic/Pathogenic Penicillium to theNewly-Identified Species

An extensive literature search was performed to identify Penicilliumspecies and organisms in related genera that are known to producetoxins. Many toxins are known to be produced by more than one species.As for all chemicals, the dose of the toxin will determine how harmfulit is to an organism. Therefore, we focused primarily on toxic compoundsthat are commonly known to cause illness in humans and other animalsfollowing consumption of foods contaminated with the toxin-producingorganism. Specifically, we looked at patulin, ochratoxins,roquefortines, PR toxin, penitrems, citrinin, penicillic acid, secalonicacid D, and wortmannin. FIG. 16 sets forth a cluster of organisms, somethat are most closely related to MM-P1, and others that are moredistantly related but still in the same genus. The phylogram was drawnfrom a maximum parsimony analysis. As seen in FIG. 16, species thatproduce these toxins are typically phylogenetically related. None of thespecies most-closely related to MM-P1 are known to produce any of thesetoxins. Indeed, P. pimiteouiense isolated from human kidney cells wasspecifically found to not produce ochratoxin A (Peterson S W et al. 1999Penicillium pimiteouiense: A New Species Isolated from Polycystic KidneyCell Cultures. Mycologia 91:269-277, the contents of which are herebyincorporated by reference in their entirety).

Also shown in FIG. 16 are some strains (e.g. P. decumbens) that wereincluded to put MM-P1 in its larger context, because they occurred in aprevious dendrogram (FIG. 15) with MM-P1; P. marneffei is includedbecause it causes infections in immune-compromised humans and P.camemberti is included because of its importance in the food industry.In cases where ribosomal RNA gene sequence could not be found (i.e., forP. confertum, P. palitans, and P. nordicum), a close relative was usedto construct the tree; the toxin occurrence indicator dot is not for thestand-in strain, except for the occurrence of ochratoxins and citrininin P. verrucosum (ochratoxins but not, to our knowledge, citrinin alsooccur in P. nordicum). The phylogenetic tree was created using Geneious(v.5.0) Tree Builder.

Example 4 Growth and Culture of MM-P1

Overview: MM-P1 is grown as a submerged, stirred culture in an aqueousmedium. Following growth, the mycelia mass is removed from the cultureby filtration and then air dried. When dry, the mycelia are subjected tosolvent extraction to remove the majority of the cellular lipids. Theremaining components of the mycelia (proteins, carbohydrates, etc.) arethen re-dried and available for the use proposed herein.

Details of culture: The substrate for growth of the culture is one ormore of the agricultural products in the following exemplary,non-exhaustive list:

-   -   Evaporated cane juice, composed primarily of sucrose but        possibly containing dextrose, fructose, cellulosic components of        sugar cane, and soil remaining from sugar cane harvest.    -   Sorghum grain    -   Water-based extract of almond hulls, possibly containing ground        almond hulls    -   Grape pomace (grape skins, seeds, and stems remaining after        juice extraction by the wine and fruit juice industries)

In addition, the following organic and inorganic (mineral) nutrients areadded to the culture medium to promote growth:

-   -   Glycerol    -   Yeast extract (water-soluble portion of autolyzed cultured        yeast) and/or corn    -   steep liquor    -   Ammonium phosphate    -   Magnesium sulfate    -   Calcium chloride    -   Ferric citrate    -   Potassium sulfate    -   Sodium acetate    -   Sodium molybdate    -   Copper sulfate    -   Cobalt nitrate    -   Zinc sulfate    -   Boric acid    -   Manganese chloride

The culture medium is sterilized using a high temperature, high pressureprotocol. After the medium has cooled, it is inoculated with MM-P1 andoxygenated. Over the course of the growth of MM-P1 (˜6 days), theculture pH is kept at or above pH 5.5 by the addition of potassiumhydroxide as needed. At the end of growth, the fungal mycelia and othersolids are separated from the culture by filtration and dried. When dry,the lipids are extracted from the mycelia using a hexane/ethanol solventmixture. The hexane/ethanol/lipid mixture is removed by decanting andthe residual solvents are then evaporated, leaving the mycelia as a drypowder.

The solid product has the following basic properties (properties mayvary as a function of feed ingredient and culture conditions):

Crude protein 26.9% Carbohydrate 30.05% Crude fiber 30.2% Crude fat1.75% Moisture 8.11% Ash 2.5%

Proteins are composed of amino acids. The amino acid composition of theproduct is as follows (properties may vary as a function of feedingredient and culture conditions):

L-Aspartic Acid 2.15% L-Threonine 1.29% L-Serine 1.51% L-Glutamic Acid5.09% Glycine 2.73% L-Alanine 4.38% L-Valine 2.15% L-Cysteine 0.07%L-Methionine 0.29% L-Isoleucine 0.79% L-Leucine 2.15% L-Tyrosine 0.36%L-Phenylalanine 0.50% L-Lysine 1.22% L-Histidine 0.14% L-Arginine 0.79%L-Proline 1.29%

Example 5 Culture of MM-P1 in a Bioprocess Reactor

MM-P1 is grown in a bioprocess reactor as a submerged, stirred culturein an aqueous medium as follows. A liquid medium in a bioprocess reactoris prepared. The liquid medium contains a carbon source at aconcentration of 15.8 g of carbon (atom) per liter. The liquid mediumadditionally contains yeast extract which comprises additional carbon.The ratio of carbon to nitrogen in the liquid medium is in the range ofabout 20:1 to about 100:1. Mineral additives as set forth in the tablebelow are added to the liquid medium. Half of the specified amount ofeach mineral in the table is added initially, and the rest is added as aconcentrated solution by sterile transfer 48 hours after onset ofgrowth. Additionally, Tween80 is added.

Molecular weight of Ingredient molecule Yeast Extract Formula (g/mol)Magnesium Sulfate MgSO₄•7H₂0 246.48 Boric acid H₃BO₃ 61.83 Manganesechloride MnCl₂•4H₂O 197.91 Zinc sulfate ZnSO₄•7H₂O 287.54 Sodiummolybdate Na₂MoO₄•2H₂O 241.97 Copper sulfate CuSO₄•5H₂O 249.68 Cobaltnitrate Co(NO₃)₂•6H₂O 291.03 Sodium Acetate C₂H₃NaO₂•3H₂O 136.08Potassium Sulfate K₂SO₄ 174.25 Calcium Chloride CaCl₂•2H₂O 147.01 FerricCitrate C₆H₈O₇Fe•xH₂O 244.95 Ammonium (NH₄)₂HPO₄ 132.06 Phosphate

The liquid culture medium is then sterilized by high temperature(121-124° C.), high pressure, (15-17 PSI). The medium is then cooled to37° C.

The sterile culture medium is then inoculated with spores (˜500 to 1000viable spores per ml final concentration). The culture is grown at36-39° C. with a sterile oxygen-enriched sparge (˜90 liters per minuteof 80% oxygen) and agitation at a stir rate of 60 RPM with a marineblade type impeller. The pH is maintained at 5.5 or above by addition ofsterile potassium hydroxide using feedback-controlled automated baseaddition.

After 48 hours of growth, the remaining mineral additives are added tothe culture. After 96 hours of growth, MgSO₄ is added to obtain a finalconcentration of 50 mM.

Following 6 days of culture, the organism is harvested by separating outthe biomass from the conditioned culture medium.

Example 6 Animal Meal Tailored for Prawns and Other Aquaculture Species

Fungal biomass can function as an animal feed by itself, but itsproperties can be enhanced by combining it with other nutrition sources.One example is provided in the case of prawn aquaculture, where thefungal biomass has been combined with other material to improve prawnsurvivability and weight gain. The following Table lists a variety ofcompositions that have been developed with fungal biomass and tested onPacific white shrimp, which is actually a prawn, Litopenaeus vannamei.

Biomeal Incorporated into Prawn Feed

B C D E F G Ingredient % % % % % % Biomeal 0.00 3.00 6.00 9.00 12.0015.00 Soybean meal, 45% 31.43 32.22 33.01 33.81 34.60 34.32 Wheat flour25.00 25.00 25.00 25.00 25.00 25.00 Feed peas 18.15 14.39 10.64 6.893.14 0.00 Fishmeal, Herring 10.00 10.00 10.00 10.00 10.00 10.00 Wheatgluten 5.00 5.00 5.00 5.00 5.00 5.00 Squid meal 2.50 2.50 2.50 2.50 2.502.50 Krill 2.50 2.50 2.50 2.50 2.50 2.50 Fish oil, 999 2.44 2.43 2.432.43 2.42 2.38 Lecithin, Deoiled 1.46 1.46 1.46 1.46 1.46 1.44 Monocalcium phosphate 1.28 1.25 1.22 1.18 1.15 1.09 Rovimix 2050 (Vitamin0.20 0.20 0.20 0.20 0.20 0.20 Mineral Premix) Cholesterol 0.05 0.05 0.050.04 0.04 0.04 All diets, above, have 35% crude protein and 6.5-6.7%crude fat, with increasing crude fiber l to r (3.1 to 6.3%)

The Table above lists the fungal biomass as “Biomeal”. A range ofcompositions was studied, ranging from zero to 15% fungal biomass, whereit was used to progressively substitute for feed pea meal, which is astandard shrimp and prawn feed ingredient. The substitution was used tomaintain protein and fat content within industry standardspecifications.

The feed formulations of the Table above were fed to batches of Pacificwhite shrimp in separate tanks, in batches of 35 prawns per tank. Prawnswere periodically removed from the tank and weighed, and mortality wasmonitored, over a six-week trial period. One tank of prawns was fed apremium commercial feed (designated as “control CP” in the Table below).Weight gain and survival was measured for all formulations and ispresented in the Table below. In the table below, “SGR” refers tospecific growth rate, and “ADG” refers to average daily weight gain.

Final Weight weight Initial weight week 3 week 6 Weight gain SGR ADGSurvival rate Diets (g) (g) (g) (%) (%/day) (g/day) (%) Control CP 1.35± 0.23 a 2.55 ± 1.05 a 4.66 ± 2.41 a 245.13 ± 28.48 a 2.94 ± 0.20 a 0.08± 0.01 a 80.00 ± 6.17 b BM 0% 1.35 ± 0.22 a 2.34 ± 0.78 a 3.61 ± 1.47 b169.00 ± 21.62 b 2.35 ± 0.19 b 0.05 ± 0.01 b 80.00 ± 4.67 b BM 3% 1.35 ±0.22 a 2.47 ± 0.77 a 3.91 ± 1.40 b 190.61 ± 16.53 b 2.54 ± 0.13 b 0.06 ±0.01 b  84.29 ± 5.95 ab BM 6% 1.35 ± 0.22 a 2.44 ± 0.77 a 4.03 ± 1.53 b202.64 ± 31.67 b 2.63 ± 0.25 b 0.06 ± 0.01 b 80.00 ± 7.00 b BM 9% 1.35 ±0.23 a 2.46 ± 0.79 a 3.87 ± 1.64 b 187.94 ± 32.81 b 2.51 ± 0.26 b 0.06 ±0.01 b 89.29 ± 3.60 a BM 12% 1.35 ± 0.22 a 2.50 ± 0.71 a 3.99 ± 1.36 b197.73 ± 17.49 b 2.59 ± 0.14 b 0.06 ± 0.01 b 90.00 ± 5.47 a BM 15% 1.35± 0.23 a 2.38 ± 0.71 a 3.77 ± 1.36 b 180.75 ± 20.47 b 2.45 ± 0.18 b 0.06± 0.01 b  85.71 ± 2.33 ab

The designations “a” and “b” in the above Table indicate outcomes thatare different in a statistically significant manner. The designation“ab” indicates an outcome that is not statistically distinguishable fromeither outcome “a” or outcome “b”. Data were analyzed using Analysis ofVariance (ANOVA) and Duncan's New Multiple Range Tests to determinewhether significant differences existed between population means. Allstatistical analyses were conducted using SPSS for Windows, version15.0. The Table shows that the prawn weight gain from samples B throughG was statistically distinguishable from that of the commercial feed,being less. This is likely attributable to the fact that the commercialfeed was available in a range of pellet sizes, small sizes being moreeasily consumed by smaller prawns earlier in the experiment, while theexperimental samples B through G were only made in a single, large,size. The larger pellets being less easily consumed by the youngerprawns accounts for the difference in weight gain, so that a repeatedtest with properly sized pellets will eliminate the difference in weightoutcome.

The results for survival rate are, however, different. In this case,samples E and F exhibited a higher survival rate that is statisticallysignificant, compared to both the commercial feed and the formulationsof samples B and D. This is considered by aquaculture experts to behighly unusual and significant, and indicates that feed formulationsincorporating the fungal biomass are, in fact, superior to existingpremium feeds.

The prawn example presented above is intended only to be exemplary andnot as limiting the detailed formulations of feeds incorporating fungalbiomass, nor the range of aquatic or terrestrial animals for which itmay be used.

Example 7 Mixture of Whey with MM-P1 Fungal-Cell Biomeal

Overview: an experiment has verified that mixing whey with fungal-cellbiomeal yields an animal feed with good nutritional properties that canbe pumped as a fluid slurry.

Dried fungal cells, after extraction of TAG, measured 1 kg. Theirdietary composition was essentially similar to the cells of Example 2.They were combined with 4 liters of whey provided by Cantaré Foods, acheese producer based in San Diego, Calif. The components of the wheywere measured to be as given in the following Table.

Cantaré Foods Whey Sample Component As sent Dry wt. Moisture (vacuumoven) 70 C. (%) 94.48 ///// Dry Matter (%) 5.52 ///// Crude Protein (%)0.4 7.32 Phosphorus (%) 0.04 0.67 Calcium (%) 0.04 0.72 Salt-NaCl (%)0.28 5.08 Chloride (%) 0.17 3.08 Glucose (%) <0.1 Fructose (% sugar)<0.1 Sucrose (% sugar) <0.1 Maltose (% sugar) <0.1 Lactose (% sugar) 4.4

The general composition of whey is provided in the following Table.

General Composition of Fresh Whey S. No Constituent Unit Sweet whey Acidwhey 1 Water % 93-94 94-95 2 Dry matter %  6-6.5 5-6 3 Lactose % 4.4-5 3.8-4.4 4 Lactic acid % traces up to 0.8 5 Total protein % 0.8-1.00.8-1.0 6 Whey protein %  0.6-0.65  0.6-0.65 7 Citric acid % 0.1 0.1 8Minerals % 0.5-0.7 0.5-0.7 9 pH 6.4-6.2 5.0-4.6 10 SH Value about 420-25

The resulting mixture contained nutritional components listed in thefollowing Table. The mixture proved easy to pour and pump.

Whey Sample - Biomeal Component As sent Dry wt. Moisture (vacuum oven)70 C. (%) 74.36 ///// Dry Matter (%) 25.64 ///// Crude Protein (%) 3.2412.6 Acid hydrolysis fat (%) 2.61 10.2 Acid detergent fiber (%) 2.9611.6 Ash (%) 1.65 6.44 Total digestible nutrients (%) 23 89.6 Netenergy-lactation (Mcal/lb) 0.24 0.94 Net energy-maint. (Mcal/lb) 0.250.99 Net energy-gain (Mcal/lb) 0.17 0.65 Digestible energy (Mcal/lb)0.46 1.79 Metabolizable energy (Mcal/lb) 0.43 1.67

The following table describes the nutrient composition in variousbiomeal:whey combinations.

Whey Sample - Biomeal Component Whey Sample 1:3 1:5 1:7 1:10 1:7(Biomeal:hot alone (Biomeal:Whey) (Biomeal:Whey) (Biomeal:Whey)(Biomeal:Whey) Whey, 70° C.) Moisture (vacuum As As As As As As oven) 70C. (%) sent Dry wt. sent Dry wt. sent Dry wt. sent Dry wt. sent Dry wt.sent Dry wt. Dry Matter (%) 94.48 ///// 74.36 ///// 80.57 ///// 83.37///// 86.81 ///// 82.77 ///// Crude Protein (%) 5.52 ///// 25.64 /////19.43 ///// 16.63 ///// 13.19 ///// 17.23 ///// Acid hydrolysis fat 0.47.32 3.24 12.6 2.23 11.5 1.78 10.7 1.26 9.58 2.1 12.2 (%) Acid detergent0.04 0.67 2.61 10.2 2.76 14.2 1.76 10.6 2.05 15.6 1.76 10.2 fiber (%)Ash (%) 0.04 0.72 2.96 11.6 3.7 19.1 2.86 17.2 3.89 29.5 3.14 18.2 Totaldigestible 0.28 5.08 1.65 6.44 0.97 4.98 0.62 3.72 0.61 4.66 0.87 5.07nutrients (%) Net energy- 0.17 3.08 23 89.6 18.1 93.1 15.1 91 12.1 91.915.3 88.8 lactation (Mcal/lb) Net energy-maint. <0.1 0.24 0.94 0.19 0.980.16 0.96 0.13 0.97 0.16 0.93 (Mcal/lb) Net energy-gain <0.1 0.25 0.990.2 1.03 0.17 1.01 0.13 1.02 0.17 0.98 (Mcal/lb) Digestible energy <0.10.17 0.65 0.13 0.68 0.11 0.66 0.09 0.67 0.11 0.65 (Mcal/lb)Metabolizable <0.1 0.46 1.79 0.36 1.86 0.3 1.82 0.24 1.84 0.31 1.78energy (Mcal/lb) Moisture (vacuum 4.4 0.43 1.67 0.34 1.74 0.28 1.71 0.231.73 0.29 1.66 oven) 70 C. (%)

Example 8 Mixture of Whey with MM-P1 Fungal-Cell Biomeal for DairyCattle

Dried MM-P1 fungal cells, after extraction of TAG, are combined withwhey provided a cheese producer, in a ratio of 1 L fungal biomass to 4 Lwhey, forming a slurry. The slurry is pumped into an empty milk tankertruck. The tanker truck transports the slurry to a dairy farm, where theslurry is pumped to a storage tank for animal feeding. Dairy cattle atthe farm are fed the slurry. The empty tanker truck is ready to befilled with milk for the return truck to the cheese producer.

Example 9 Inhibition of Gram-Positive Bacterium by Extracts from MM-P1Culture Medium

This example describes the unexpected discovery that MM-P1 fungal cellsproduce an antibiotic substance that inhibits the growth ofgram-positive bacterium Bacillus subtilis. MM-P1 was cultured in aliquid medium. The fungal culture was filtered to separate the fungalcells from the conditioned culture medium. The conditioned culturemedium was then concentrated by drying. The resultant residue wassolubilized in sterile water. Any remaining water-insoluble residue wasthen contacted again with water.

An agar plate with bacterial growth medium was inoculated with B.subtilis and incubated under standard growth conditions. Discs soaked intest compositions were than added to the resulting lawn of bacteria andthe plate was incubated for an additional period to test for inhibitionof growth.

As seen in FIG. 17, a zone of inhibition was detected for thenon-water-soluble portion of the extract, but not for a control sampleof water. The inhibitory compound appeared concentrated in the non-watersoluble (i.e., solid or not easily dissolved in water) portion of theextract. These results indicated that MM-P1 culture medium contains asubstance capable of inhibiting the growth of gram-negative B. subtilis.

Example 10 Inhibition of Gram-Positive Bacterium by Extracts from MM-P1Culture Medium

This example describes further extraction and isolation of one or moreinhibitory substances produced by MM-P1 fungal cells that inhibits thegrowth of gram-positive bacteria. A variety of feedstocks were tested inthe liquid culture medium, in order to determine whether the type ofcarbon source had an effect on the level of production of inhibitorysubstances. The liquid culture medium conditioned by growth of MM-P1 wasfirst filtered to separate the medium from the fungal cells.

As set forth in FIG. 18, a five liter volume of each type of liquidmedium was then concentrated to a volume of 300 mL by removal of themajor portion of water. The concentrated solution was then subjected tosolvent extraction, using a series of solvents with increasing polarity.The solvent extractions were sequential for each filtrate batch. Forexample, as set forth in the table below, MAA-182-11-3 is the amylacetate extract of the mixture remaining after extraction with a mixtureof methyl t-butyl ether and methylene chloride. Extraction with hexanedid not yield any detectable material. An aliquot of each extract wasthen tested at 1000 μg/ml and at 500 μg/ml for inhibition of growthagainst both Gram-positive and Gram-negative bacteria. The table belowsets forth the surprising results of the test.

TABLE Effectiveness of extracts against Gram negative and Gram-positivebacteria Effectiveness Effectiveness against Gram- against Gram-Compound negative positive number Solvent for extraction bacterium*bacterium* Feedstock of Culture MAA-182-10-3 Ethyl acetate X + + + + +Sorghum stalk and glycerol in MAA-182-10-4 20% isopropanol in X + + +recycled (x2) sorghum stalk methylene chloride and glycerol brothMMA-182-10-5 Ethyl acetate at pH 2.5 X + + MAA-182-10-6 Combined methylt butyl X + + ether and methylene chloride extractions MAA-182-11-1Methyl t butyl ether X X Almond hulls in recycled (x2) MAA-182-11-2Methylene chloride X + + + + almond hull broth MAA-182-11-3 Amyl acetateX + + + MAA-182-11-4 20% isopropanol in X + + + + + methylene chlorideMAA-182-11-5 Amyl acetate at pH 2.5 X + + + + MAA-182-12 20% isopropanolin + + + + + + + + + Sorghum stalk in recycled methylene chloride (x1)sorghum stalk broth MAA-182-13-1 Methyl t butyl ether X + + + + +Sorghum stalk in recycled MAA-182-13-2 Amyl acetate X X (x1) sorghumstalk broth MAA-182-13-3 20% isopropanol in X X methylene chloride *X =no effect; + to + + + + + indicates degree of effectiveness (increasing)

As demonstrated by the results set forth in the Table above, at leastfour of the extracts tested had the highest possible observedeffectiveness against Gram-positive bacterial growth. At least two ofthe extracts tested had the next-highest observed effectiveness.Further, at least one of the extracts tested (MAA-182-12) was effectiveagainst Gram-negative bacterial growth. These results demonstrate thatMM-P1 produces a broad spectrum antibacterial substance that is capableof concentration and extraction.

The above description of the disclosed embodiments is provided to enableany person skilled in the art to make or use the invention. Variousmodifications to these embodiments will be readily apparent to thoseskilled in the art, and the generic principles described herein can beapplied to other embodiments without departing from the spirit or scopeof the invention. For example, while the feedstock received by acellulose processing plant has been referred to as containing cellulosicmaterial, any type of feedstock which may yield alkanes and/or aromaticcompounds may be used. Thus, it is to be understood that the descriptionand drawings presented herein represent a presently preferred embodimentof the invention and are therefore representative of the subject matterwhich is broadly contemplated by the present invention. It is furtherunderstood that the scope of the present invention fully encompassesother embodiments that may become obvious to those skilled in the artand that the scope of the present invention is accordingly limited bynothing other than the appended claims.

1. A conditioned medium comprising an inhibitory substance, wherein saidmedium is conditioned by a cellulose degrading fungus.
 2. Theconditioned medium of claim 1, wherein said cellulose degrading fungusis a fungus of the genus Penicillium.
 3. The conditioned medium of claim1, wherein said cellulose degrading fungus is a lignocellulose degradingfungus.
 4. The conditioned medium of claim 1, wherein said cellulosedegrading fungus comprises a characteristic selected from the groupconsisting of: a 5.8 S ribosomal RNA gene sequence having at least 98%nucleotide sequencing identity with the nucleic acid of SEQ ID NO: 1; anITS1 sequence with at least 98% sequence identity to SEQ ID NO: 2; anITS2 sequence with at least 98% sequence identity to SEQ ID NO: 3; and a28 S ribosomal RNA gene sequence with at least 98% sequence identity toSEQ ID NO:
 4. 5. The conditioned medium of claim 1, wherein saidcellulose degrading fungus comprises a species selected from the groupconsisting of: Penicillium menonorum and a species being the same asNRRL deposit Accession No:
 50410. 6. (canceled)
 7. (canceled) 8.(canceled)
 9. A process for producing an inhibitory substancecomprising: a) incubating a cellulose degrading fungus of the genusPenicillium in a liquid medium for a sufficient time for production ofan inhibitory substance by said fungus; and b) separating said fungusfrom said liquid medium, thereby producing a harvested fungus and aconditioned liquid medium, wherein said conditioned liquid mediumcomprises said inhibitory substance produced by said fungus.
 10. Theprocess of claim 9, wherein said cellulose degrading fungus is alignocellulose degrading fungus.
 11. (canceled)
 12. (canceled) 13.(canceled)
 14. (canceled)
 15. (canceled)
 16. The process of claim 9,further comprising: c) extracting said inhibitory substance from saidconditioned medium.
 17. A conditioned medium containing an inhibitorysubstance, said conditioned medium produced by the process of claim 9.18. An extract comprising an inhibitory substance, said extract producedby the process of claim
 16. 19. A vessel comprising an antimicrobialsubstance obtained from a cellulose degrading fungus of the genusPenicillium.
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)24. (canceled)
 25. (canceled)
 26. An extract of medium conditioned by acellulose degrading fungus of the genus Penicillium, said extractcomprising one or more extraction-soluble compounds having antimicrobialactivity.
 27. The extract of claim 26, wherein said extract comprisesone or more hexane soluble compounds.
 28. The extract of claim 26,wherein said extract comprises one or more methy t-butyl ether solublecompounds.
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled)